sRNA centered signaling activates nitrate respiration and enhances Cronobacter sakazakii virulence in host environments

Domain: chemistry
Published: 2026-03-03
Editorial outcome: major_revision

Abstract
Cronobacter sakazakii is an important neonatal pathogen frequently associated with
powdered infant formula. However, the mechanisms by which C. sakazakii adapts to the host
environment and establishes systemic dissemination remain poorly understood. Here, we reveal a
signal transduction pathway centered on a novel sRNA, CsrN, which facilitates C. sakazakii in
utilizing nitrate respiration in response to oxygen-limited environments within the host, thereby
enhancing its virulence in vivo. C. sakazakii infection triggers an inflammatory response, leading
to the accumulation of host-derived nitrate, a key alternative electron acceptor. The expression of
CsrN is induced under anaerobic conditions via the ArcAB two-component regulation system.
CsrN subsequently enhances the expression of the narGHJI operon, which encodes a nitrate
reductase complex. This promotes the colonization of C. sakazakii in the gastrointestinal tract
and benefits its survival within macrophages, ultimately leading to increased systemic bacterial
dissemination and virulence in the host. We show that administration of tungstate, a specific
inhibitor of nitrate respiration, significantly attenuates C. sakazakii virulence in animal
experiments. This work provides novel insights into the survival and pathogenicity mechanisms
employed by C. sakazakii in host environments and suggests nitrate respiration as a potential
therapeutic target for combating C. sakazakii infections.

Introduction
Cronobacter spp, are Gram-negative, facultatively anaerobic bacteria recognized as
opportunistic pathogens that pose a serious threat to neonates and immunocompromised infants
1,2. Among them, Cronobacter sakazakii is the most clinically significant species, frequently
associated with both outbreaks and sporadic cases worldwide 3. Infection caused by C. sakazakii
can result in life-threatening conditions, such as bacteremia, sepsis, necrotizing enterocolitis,
meningitis and brain abscesses, with reported mortality rate reaching up to 80% based on
epidemiological data 4,5. Survivors often suffer from long-term neurological complications,
including quadriplegia, hydrocephalus and delayed neural development 6. Although C. sakazakii
is ubiquitous in the environment, its primary transmission source is reconstituted powdered
infant formula (PIF) 7. This is largely attributed to the bacterium’s remarkable resistance to
desiccation, osmotic stress, and high temperature 1,2,6,8. Furthermore, the emergence of multi-
drug resistant C. sakazakii renders the effective treatment more challenging, which has been
regarded as an important concern in public health 9.

Beyond reducing contamination risks during PIF production, a comprehensive
understanding of the virulence mechanisms of C. sakazakii is essential for developing effective
preventive and therapeutic strategies. The infection process of C. sakazakii typically begins with
the colonization of the gastrointestinal tract (GIT) 2. Several virulence factors contribute to the
ability of C. sakazakii to adhere to intestinal mucosal surfaces, among which OmpA and OmpX
play particularly prominent roles 7. Following colonization, C. sakazakii can translocate into the
bloodstream, where it exhibits systemic persistence by evading immune responses and
replicating within macrophages 7,10,11. Survival within macrophages is an essential step for C.
sakazakii to establish systemic dissemination, as the pathogen can evade the host immune
defense, penetrate the tissue barrier and translocate to the bloodstream, liver, and spleen through
macrophages 2,7,12. Furthermore, C. sakazakii is capable of penetrating the blood-brain barrier
(BBB) by invading human brain microvascular endothelial cells, ultimately leading to meningitis
7,13.

Adenosine triphosphate (ATP) is essential for bacterial proliferation, serving as the primary
energy source for biosynthetic processes. Bacterial cells generate ATP through a series of redox
reactions, whereby electrons are transferred from electron donors, such as glucose, to electron
acceptors, with oxygen (O2) being the most efficient due to its high redox potential. This process
releases substantial free energy that fuels cellular activities 14. However, within host
environments, bacteria frequently encounters oxygen-limited conditions, which necessitate the
use of alternative electron acceptors, such as tetrathionate, nitrate and fumarate 15,16. The ability
to efficiently exploit these alternative respiratory pathways enable pathogens to maximize energy
production, thereby facilitating rapid proliferation and competitive advantage over commensal
microbes 14. However, the mechanisms by which C. sakazakii adapts to the gut environment and
establishes systemic dissemination remain largely unclear.

The process of C. sakazakii infection is shaped by a complex array of host environment
factors. In bacteria, small RNAs (sRNAs) play crucial roles in regulating gene expression in
response to changing environmental conditions 17. A critical regulatory mechanism employed by
many pathogens involves post-transcriptional modulation via sRNAs, which function by base-
pairing with specific mRNA targets to influence processes such as growth, metabolism, cell
signaling, and immune evasion 17-19. Despite their recognized importance in other bacterial
pathogens, the specific roles of sRNA in C. sakazakii virulence remain largely unexplored.

In this study, we identified a signal transduction pathway that activates nitrate respiration,
thereby enhancing the pathogenicity of C. sakazakii through an sRNA, CsrN. We found that the
expression of CsrN is induced by the ArcAB two-component systems under anaerobic condition.
CsrN increases the expression of narGHJI, which encodes the nitrate reductase, by directly
binding to the 5′ untranslated region (UTR) of narGHJI mRNA. Activation of nitrate respiration
significantly promotes the survival of C. sakazakii in both the gut lumen and macrophages,
ultimately facilitating its systemic dissemination within the host. Furthermore, administration of
tungsten to neonatal rats significantly attenuated gastrointestinal colonization and systemic
dissemination of C. sakazakii by inhibiting the molybdenum-cofactor-dependent nitrate
respiration pathway. Our results suggest that targeting nitrate respiration may represent a
promising therapeutic strategy for combating C. sakazakii infection.

Results
CsrN is a small RNA in C. sakazakii that is induced during infection
Using publicly available RNA-seq data from C. sakazakii ATCC 29544 during its infection
of RAW264.7 cells 20, we identified a previously uncharacterized sRNA located in the intergenic
region between the genes yhcB (inner membrane protein) and degQ (HtrA family serine
endoproteases), and named it CsrN (Fig. 1A). Notably, the expression of CsrN was found to be
upregulated during infection 20, indicating a potential role in the virulence of C. sakazakii (Fig.
1B).

To determine whether CsrN functions as an sRNA in C. sakazakii ATCC 29544, we
conducted Northern blotting using a CsrN-specific probe. A distinct RNA band was detected in
both the wild-type (WT) and the ΔCsrN complemented strain (ΔCsrN+pCsrN), but was absent in
the ΔCsrN strain, indicating that CsrN is transcribed from the reverse strand of the C. sakazakii
ATCC 29544 genome (Fig. 1C). To precisely map the ends of CsrN, we employed 5′ and 3′ rapid
amplification of cDNA ends (RACE), which confirmed that CsrN was 181 nt in length, spanning
coordinates 608,538 to 608,718 in the genome (Supplementary Fig. 1A). Secondary structure
prediction using RNAfold 21 revealed that CsrN consists of six hairpin loops, five interior loops
and one multiloop (Fig. 1D).

Given that the RNA chaperone Hfq is often involved in sRNA stability and function 22, we
investigated whether CsrN requires Hfq. Northern blotting analysis revealed no significant
difference in CsrN expression between Δhfq and WT strains (Supplementary Fig. 1B).
Additionally, a rifampicin transcription inhibition assay 22 showed that the degradation rates of
CsrN exhibited no significant difference between Δhfq and WT strains, indicating that Hfq does
not influence the stability of CsrN (Supplementary Fig. 1C). These results suggest that CsrN is
an Hfq-independent sRNA.

A sequence homology search showed that CsrN is present only in Cronobacter spp. In order
to explore the distribution of CsrN in C. sakazakii, we performed a comparative genomics
analysis using 188 publicly available complete C. sakazakii genomes (Supplementary Data 1).
The results showed that CsrN was present in 97.3% C. sakazakii strains (183/188), suggesting
the prevalence and importance of CsrN in C. sakazakii.

CsrN enhances C. sakazakii virulence in vitro and in vivo
Given the replication within macrophages enables pathogens to establish systemic disease,
we infected RAW264.7 cells with C. sakazakii to assess whether CsrN contributes the survival of
C. sakazakii in macrophages. Growth curves analysis showed that WT and ΔCsrN exhibited
similar growth rates in monoculture in vitro (Supplementary Fig. 2A-B). However, the survival
of CsrN within RAW264.7 cells was significantly decreased compared to that of WT and
ΔCsrN complemented strains at 1, 4, 24 h post infection (hpi) (Fig. 2A). These results indicate
that CsrN promotes the intracellular survival of C. sakazakii in macrophages. Meanwhile, we
also detected the cytotoxicity in macrophages infected with WT, ΔCsrN or ΔCsrN
complemented strain, as measured by LDH release at 1, 4, 24 hpi. The result showed that the
ΔCsrN strain was significantly less cytotoxic to macrophages compared to WT strain
(Supplementary Fig. 2C). These results indicate that deletion of CsrN in C. sakazakii reduces
bacterial intracellular survival and cytotoxicity in macrophages.

To investigate the role of CsrN in bacterial pathogenicity in vivo, we orally infected 2- to 5-
day-old rats with WT, CsrN and ΔCsrN complemented strains. Specimens from the small
intestine, colon, blood, liver, and spleen were collected at 48 hpi and plated on the LB agar plate
to assess GIT colonization and systemic dissemination. The results showed that rats infected with
CsrN exhibited significant lower bacterial burdens in the small intestine, colon, blood, liver,
and spleen compared to those infected with WT and ΔCsrN complemented strains, suggesting
that CsrN contributes to both GIT colonization and systemic dissemination of C. sakazakii within
the host (Fig. 2B). Furthermore, rats infected with CsrN exhibited prolonged survival times
compared to those infected with WT and ΔCsrN complemented strains (Fig. 2C). To further
validate these findings, we constructed a stable luciferase reporter plasmid using the frr promotor
and luxCDABE operon, following the method described by Kevin Howe 23, and transformed it
into WT, CsrN, and ΔCsrN complemented strains, respectively. This allowed us to monitor
infection level in rats using live imaging. Bioluminescence imaging (BLI) showed that rats
infected with CsrN-Lux exhibited significantly reduced dissemination of bacteria into systemic
organs compared with those infected with WT-Lux or ΔCsrN complemented-Lux strain (Fig.
2D). We further investigated the inflammatory factor production and histological damage in the
intestines of neonatal rats infected by C. sakazakii. The results showed that the levels of IL-6 and
TNF-α, as well as histopathology scores, were significantly higher in WT-infected rats than those
in CsrN-infected rats (Fig. 2E-F and Supplementary Fig. 2D), indicating that the severity of
disease caused by ΔCsrN infection in neonatal rats was significantly decreased compared to that
caused by WT infection. Collectively, these results indicate that CsrN plays a critical role in the
virulence of C. sakazakii in vivo.

CsrN enhances narGHJI expression by stabilizing its mRNA
To investigate the molecular mechanism by which CsrN regulates the virulence of C.
sakazakii, we compared the transcriptional profiles of WT and ΔCsrN using RNA-seq. A total of
124 differentially expressed genes between the two strains were identified, including 29 genes
that were significantly down-regulated and 95 genes that were up-regulated in the absence of
CsrN (Fig. 3A, Supplementary Data 2).

KEGG pathway enrichment analysis revealed distinct regulatory patterns in the ΔCsrN
strain, with the top five significantly up- and down-regulated pathways further visualized
through chord diagrams (Fig. 3B). Based on RNA-seq results, we found that the expression of
narGHJI operon (Fig. 3C), which encodes a membrane-bound anaerobic nitrate reductase
complex 24 related to nitrogen metabolism in KENGG pathways, was significantly down-regulated
in ΔCsrN (Fig. 3A-B).

The nitrate reductase complex functions to reduce nitrate to nitrite, a key process in
anaerobic nitrate respiration 14. To confirm that narGHJI is regulated by CsrN, we performed
qRT-PCR analysis on WT and ΔCsrN grown in LB medium. The results showed a significant
reduction in narGHJI expression in ΔCsrN compared to WT strain (Fig. 3D), consistent with our
RNA-seq data (Fig. 3A-B). Additionally, during macrophage infection, the expression of
narGHJI in ΔCsrN was also significantly reduced at 1, 4, 24 hpi (Fig. 3E and Supplementary
Fig. 3A-B). A similar down-regulation trend was also observed in the small intestine and colon
of rats infected with ΔCsrN compared with those infected by WT (Fig. 3F and Supplementary
Fig. 3C). These results indicated that CsrN positively regulates the expression of narGHJI both
in vitro and in vivo.

To investigate whether CsrN directly regulates the expression of narGHJI, we analyzed
potential base-pairing interactions between CsrN and narGHJI using IntaRNA 25. The analysis
revealed a 14-nucleotide complementary region between the 5′ UTR of narGHJI mRNA
(GCGUUACCGUCCCG) and the CsrN sRNA (CGGGACGGCAGCGC), with a 3-nucleotide
motif in the CsrN hairpin loop (CGG) that may facilitate the binding (Fig. 3G). Subsequently, we
conducted RNA electrophoretic mobility shift assay (REMSA) to test whether there is a direct
interaction in vitro, using CsrN sRNA and 5′ UTR of narGHJI mRNA obtained though in vitro-
transcription. The results demonstrated that CsrN sRNA binds to the 5′ UTR of narGHJI mRNA,
as well as to its complementary strand (CsrN+, positive control), in a dose-dependent manner
(Fig. 3H and Supplementary Fig. 3D). In contrast, no interaction was observed between the
mutant CsrN sRNA (CsrNmut, with the binding site CGG mutated to AGC) and 5′ UTR of
narGHJI mRNA (Fig. 3I), or between CsrN sRNA and a mutant 5′ UTR of narGHJI mRNA (5′
UTRmut of narGHJI, with the binding site CCG mutated to GCU) (Fig. 3J). However, a direct
interaction was observed between the CsrNmut sRNA and 5′ UTRmut of narGHJI mRNA (Fig.
3K). These results indicate that CsrN increases the expression of narGHJI by directly binding to
the 5′ UTR of narGHJI mRNA through a specific binding motif in the hairpin loop.

To further confirm the regulatory role of CsrN, we constructed a luciferase reporter plasmid
by fusing the promoter and 5′ UTR of narGHJI or its mutant (5′ UTRmut of narGHJI, with CCG
mutated to GCU) with luxCDABE. These plasmids were transformed into ΔCsrN to generate
strains ΔCsrN narGHJI-lux and ΔCsrN narGHJImut-lux, respectively. Overexpression of CsrN in
ΔCsrN narGHJI-lux strain significantly increased luminescence intensity, while overexpression
of CsrNmut had no effect (Fig. 3L). In contrast, the luminescence intensity of ΔCsrN narGHJImut-
lux exhibited no significant change upon overexpression of CsrN, but exhibited a significant
increase when CsrNmut was overexpressed (Fig. 3M), confirming that CsrN increases the
expression of narGHJI by directly binding to the 5′ UTR of narGHJI mRNA.

To investigate whether CsrN increases the expression of narGHJI by influencing its mRNA
stability, we performed a rifampicin transcription inhibition assay. The results showed that
narGHJI mRNA degraded more rapidly in ΔCsrN compared to WT, as evidenced by reduced
expression of narH and narJ in ΔCsrN (Fig. 3N-O), indicating that CsrN enhances the stability
of narGHJI mRNA. Additionally, the expression of narH and narJ in Δhfq strain showed no
significant difference compared to WT strain (Supplementary Fig. 3E-F). These results
demonstrate that CsrN directly binds to the 5′ UTR of narGHJI mRNA in an Hfq-independent
manner, promoting its expression by stabilizing the mRNA.

narGHJI promotes the pathogenicity of C. sakazakii
To investigate the role of narGHJI in C. sakazakii virulence, we constructed a ΔnarGHJI
mutant strain and a double-knockout strain ΔCsrNΔnarGHJI. Growth curve analysis revealed no
significant difference in growth rates among WT, ΔnarGHJI and ΔCsrNΔnarGHJI strains when
cultured in vitro (Supplementary Fig. 4A-B). However, the survival of narGHJI in macrophage
was significantly reduced compared with that of WT (Fig. 4A). This survival deficiency was
restored to WT levels through plasmid complementation (Fig. 4A). It indicates that narGHJI
benefits the survival of C. sakazakii in macrophages. In addition, the survival of
ΔCsrNΔnarGHJI exhibited no significant difference compared with that of ΔnarGHJI or
ΔCsrNΔnarGHJI complemented with CsrN (ΔCsrNΔnarGHJI + pCsrN) strain, indicating that
CsrN deletion did not affect the bacterial survival in macrophages in a ΔnarGHJI background
(Fig. 4A and Supplementary Fig. 4C). The analysis of cytotoxicity in macrophages showed that
the ΔnarGHJI strain was significantly less cytotoxic to macrophages compared to WT strain
(Supplementary Fig. 4D). These results suggest that deletion of narGHJI in C. sakazakii reduces
bacterial intracellular survival and cytotoxicity in macrophages.

To investigate whether narGHJI contributes to systemic dissemination in vivo, neonatal rats
were orally infected with the WT, narGHJI, ΔCsrNΔnarGHJI and ΔnarGHJI complemented
strains (ΔnarGHJI+pnarGHJI), respectively. The results showed that rats infected with
narGHJI exhibited significantly lower bacterial burdens in the blood, liver, and spleen
compared with those infected with WT or ΔnarGHJI complemented strain (Fig. 4B). We also
evaluated the colonization of ΔnarGHJI in the small intestine and colon, an environment with
limited oxygen 14. The results revealed that narGHJI was essential for the colonization of C.
sakazakii in the GIT, as rats infected with ΔnarGHJI exhibited a significantly decreased bacterial
burdens in the small intestine and colon compared with those infected with WT or ΔnarGHJI
complemented strain (Fig. 4B). Moreover, rats infected with narGHJI exhibited prolonged
survival times compared to those infected with WT or ΔnarGHJI complemented strain (Fig. 4C).
BLI analysis also showed that narGHJI-Lux exhibited reduced dissemination into systemic
organs compared with WT-Lux or ΔnarGHJI complemented-Lux strain (Fig. 4D). These data
indicate that narGHJI contributes to the virulence of C. sakazakii in vivo. Furthermore, no
significant differences in the survival rates were observed among rats infected with
ΔCsrNΔnarGHJI, ΔnarGHJI or ΔCsrNΔnarGHJI + pCsrN strain (Fig. 4C and Supplementary
Fig. 4E). The bacterial burdens in the blood, liver, spleen, small intestine and colon of rats
infected with ΔCsrNΔnarGHJI exhibited no significant difference from those in rats infected
with ΔnarGHJI or ΔCsrNΔnarGHJI + pCsrN strain, confirming that CsrN’s impact on C.
sakazakii pathogenicity is medicated through narGHJI (Fig. 4B and Supplementary Fig. 4F).
In
addition, we found that the levels of IL-6 and TNF-α, as well as the histopathology scores, in the
intestines of neonatal rats infected with WT were significantly higher than those of rats infected
with narGHJI (Fig. 4E-F and Supplementary Fig. 4G). Collectively, these results showed that
narGHJI plays a critical role in the pathogenicity of C. sakazakii within the host.
Moreover, we showed that the 5′ UTR mut-nar strain (mutated the binding motif of narGHJI 5′
UTR (CCG to GCU) on the chromosome of WT) and CsrNmut strain (mutated the binding motif
of CsrN (CGG to AGC) on the chromosome of WT) exhibited significantly reduced survival in
macrophage at 1, 4, 24 hpi, as well as markedly attenuated systemic dissemination levels in
neonatal rats compared to WT strain (Supplementary Fig. 4H-I). In contrast, the 5′ UTRmut-
narCsrNmut strain, in which the binding motifs in 5′ UTR of narGHJI (CCG to GCU) and CsrN
(CGG to AGC) were simultaneously mutated on the chromosome of WT, exhibited restored
bacterial survival in macrophage and in vivo bacterial burdens in neonatal rats comparable to WT
level (Supplementary Fig. 4H-I). These results further confirm that CsrN positively regulates
narGHJI expression through a specific RNA-RNA interaction mediated by its binding motif,
thereby promoting C. sakazakii survival in macrophages and facilitating systemic dissemination
in the host.

Nitrate respiration is essential for the full virulence of C. sakazakii
Previous studies have demonstrated that intestinal inflammation can create a nutrient-rich
niche that promotes bacterial replication within the host 26. For instance, Salmonella exploits
host-derived nitrate and tetrathionate generated during inflammation as alternative electron
acceptors to support anaerobic respiration, providing a metabolic advantage over other gut
microbes 16,27. To investigate whether the attenuated virulence observed in ΔnarGHJI is
associated with impaired nitrate respiration, we first measured nitrate levels in the small intestine
and colon of C. sakazakii-infected rats, as well as in the supernatant of infected macrophage
cultures at 1, 4, 24 hpi in vitro. Compared to uninfected controls, nitrate levels were significantly
elevated in both intestinal tissues and macrophage supernatants following infection (Fig.
5A-B).
Then we performed qRT-PCR to analyze the expression of host proinflammatory markers (Tnf,
Nos2) in the small intestine and colon of C. sakazakii-infected rats and in C. sakazakii-infected
macrophages at 1, 4, 24 hpi in vitro. Notably, Nos2 encodes inducible nitric oxide synthases
(iNOS), which catalyzes the production of nitric oxide (NO). NO is subsequently oxidized or
reacts with reactive oxygen species to form nitrate 28. We found that the expression of these host
proinflammatory genes was significantly increased in infected samples in vivo and in vitro (Fig.
5C-D). These results indicate that C. sakazakii infection induces an inflammatory response,
leading to elevated Nos2 expression and nitrate accumulation within hosts, which may facilitate
the growth of C. sakazakii through nitrate respiration.

NarK, a nitrate/nitrite transporter, is always highly expressed under anaerobic conditions in
the presence of nitrate and plays a central role in bacterial nitrate uptake 29. To further confirm
the role of nitrate respiration on C. sakazakii pathogenicity, we examined the expression of
narGHJI and narK by qRT-PCR in infected macrophages. The results showed that both the
expression of narGHJI and narK in C. sakazakii was significantly upregulated in infected
macrophages at 1, 4 and 24 hpi, compared with that of bacteria grown in LB medium (Fig. 5E
and Supplementary Fig. 5A-B). Then we constructed a ΔnarK mutant and showed that its
survival in macrophages was significantly decreased compared to that of WT strain (Fig. 5F).
Additionally, rats infected with ΔnarK exhibited significantly lower bacterial burdens in the
small intestine, colon, blood, liver, and spleen compared with those of WT-infected rats (Fig.
5G). These findings further underscore the essential role of nitrate respiration in C. sakazakii
survival and systemic dissemination during host infection.

Due to its high redox potential, nitrate serves as a preferred electron acceptor under
anaerobic respiration 30. To investigate its role in C. sakazakii growth, we performed bacterial
growth experiments under both aerobic and anaerobic conditions, with or without nitrate
supplementation. The results showed that the growth of ΔnarGHJI, ΔnarK, and WT exhibited no
significant difference in aerobic environments, regardless of nitrate supplementation
(Supplementary Fig. 5C). However, under anaerobic conditions, the WT strain exhibited
significantly enhanced replication compared to the ΔnarGHJI and ΔnarK at 1, 4, 24 hours (Fig.
5H), and showed slightly increased growth compared to the ΔCsrN strain, although this
difference was not statistically significant (Supplementary Fig. 5D). Moreover, nitrate
supplementation significantly promoted the anaerobic growth of both WT and ΔCsrN strains, but
had no significant effect on the growth of ΔnarGHJI and ΔnarK strains (Fig. 5H and
Supplementary Fig. 5D). Notably, under nitrate-supplemented conditions, the WT strain showed
significantly greater growth than ΔCsrN (Supplementary Fig. 5D). Infection assays in
macrophages with or without nitrate supplementation demonstrated that nitrate supplementation
significantly enhances the intracellular survival of WT and ΔCsrN strains (Fig. 5I and
Supplementary Fig. 5E), but had no effect on the survival of either ΔnarGHJI or ΔnarK (Fig. 5I).
These results indicate that C. sakazakii is capable of utilize nitrate as an electron acceptor to
support anaerobic respiration, which in turn promotes its survival and proliferation.

ArcAB senses anaerobic condition to enhance narGHJI expression via CsrN
To investigate the mechanism through which the expression of CsrN and narGHJI is
induced within host, we performed a DNA pull-down assay to identify potential regulators
binding to the promoter of CsrN. The mass spectrometry result showed that ArcA is a potential
regulator that binds to the promoter of CsrN (Supplementary Data 3). ArcA, together with ArcB,
forms a two-component regulation system (TCS) that sense changes in oxygen availability and
regulate the expression of downstream genes 31. We showed that the expression of CsrN was
significantly decreased in arcA mutant compared to WT when grown in LB medium under
anaerobic conditions, but there was no significant difference under aerobic conditions (Fig. 6A
and Supplementary Fig. 6A). Additionally, the expression of both CsrN and narGHJI was
significantly decreased in ∆arcA mutant compared to the WT strain in infected macrophages
which represent an oxygen-limited condition 33 (Fig. 6B and Supplementary Fig. 6B-C),
indicating that ArcA positively regulates CsrN and narGHJI expression under anaerobic
conditions. Moreover, no significant difference in narGHJI expression was observed between
CsrN and CsrNarcA strains under either aerobic or anaerobic conditions (Fig. 6C and
Supplementary Fig. 6D), suggesting that ArcA regulate the expression of narGHJI through CsrN.
Electrophoretic mobility shift assay (EMSA) showed that phosphorylated ArcA (but not
unphosphorylated ArcA) binds to the CsrN promoter in a dose-dependent manner (Fig. 6D). In
contrast, ArcA showed no binding to a DNA fragment derived from the kana gene (negative
control) (Supplementary Fig. 6E). Chromatin immunoprecipitation (ChIP) qPCR demonstrated
that the CsrN promoter (PCsrN) was enriched 2.78-fold in the ArcA-ChIP samples compared to
the Mock-ChIP samples (Fig. 6E). As a control, no significant enrichment was observed for
rpoS. These results indicate that ArcA directly activates the expression of CsrN by binding to its
promoter.

ArcA, a key regulator of anaerobic responses, enables bacteria to adapt to anaerobic
environments 32. qRT-PCR assays and northern blot analysis were performed to further analyze
CsrN expression in C. sakazakii cultured aerobically or anaerobically in minimal media. The
results showed a significant upregulation of CsrN expression in WT under anaerobic condition
compared to aerobic condition (Fig. 6F-G). Consistently, the expression of the narGHJI, the
downstream target of CsrN, was also significantly elevated under anaerobic conditions compared
to aerobic conditions (Fig. 6G). In contrast, no significant difference in the expression of CsrN
and narGHJI was observed in ΔarcA between aerobic and anaerobic conditions (Fig. 6H-I).
These results further confirmed that ArcA promotes the expression of CsrN and narGHJI under
anaerobic conditions. Furthermore, qRT-PCR assays showed that the CsrN and narGHJI
expression of C. sakazakii in WT-infected rats, were significantly upregulated in the small
intestine and colon, in which oxygen availability is limited, compared to that grown in LB
medium (Supplementary Fig. 6F-G), in agreement with in vitro anaerobic culture data (Fig. 6F-
G). However, the expression of CsrN and narGHJI in arcA in the small intestine and colon of
infected rats exhibited no significant difference compared to that grown in LB medium
(Supplementary Fig. 6H-I). Previous study reported that the intracellular environment is also
oxygen-limited 33, consistent with our observation of elevated expression of CsrN and narGHJI
in macrophages (Fig. 1B, 5E, and Supplementary Fig. 5A-B). In contrast, when we infected
macrophages with arcA, the expression of CsrN and narGHJI at 1, 4, 24 hpi exhibited no
significant difference compared to that of arcA grown in LB medium (Supplementary Fig. 6J-
L). Moreover, we found that the survival of ΔarcA in macrophages and its system dissemination
in vivo were significantly reduced compared to those of the WT or ΔarcA complemented strain
(Fig. 6J and Supplementary Fig. 6M). Collectively, these results demonstrate that ArcA functions
as an oxygen sensor within the host, activating CsrN expression under anaerobic conditions.
This, in turn, leads to the induction of narGHJI and promotes nitrate respiration, thereby
contributing to the pathogenicity of C. sakazakii.

Targeting nitrate respiration to control C. sakazakii infection
Bacteria exhibit remarkable metabolic versatility, enabling them to adapt to varying oxygen
levels and electron acceptor availability 34. To evaluate the role of aerobic respiration, anaerobic
respiration, and fermentation on C. sakazakii pathogenicity, we constructed mutants in several
key metabolic pathways: cydAB (encoding cytochrome bd oxidase, a terminal oxidase facilitating
aerobic respiration under low-oxygen conditions), frdABCD (encoding fumarate reductase,
which reduces fumarate as an alternative terminal electron acceptor in anaerobic respiration),
dmsABC (encoding DMSO reductases involved in DMSO respiration under anaerobic
conditions), adhE (involved in ethanol fermentation) and ldhA (involved in lactate fermentation)
strains. Growth curve analysis showed no significant difference in growth between these mutants
and WT under both aerobic (Supplementary Fig. 7A-B) and anaerobic conditions (Fig. 7A).
Intracellular survival assays showed that the deletion of cydAB, frdABCD, dmsABC, adhE or
ldhA did not influence the survival of C. sakazakii in macrophages (Fig. 7B). In addition,
infection assays in rats revealed no significant difference in bacterial burdens in the intestine or
systemic organs between the mutants and WT (Fig. 7C). These findings suggest that none of the
aerobic or anaerobic metabolic pathways, including those governed by cydAB, frdABCD,
dmsABC, adhE, and ldhA, are essential for the virulence of C. sakazakii. Instead, these results
imply that nitrate respiration is the primary metabolic pathways utilized by C. sakazakii to thrive
within the host.

Our above results showed that nitrate respiration plays a vital role in the colonization of GIT
and systemic dissemination by C. sakazakii within the host. Tungstate has been shown to disrupt
nitrate respiration in Enterobacteriaceae by substituting molybdenum in the molybdopterin
cofactor, an essential component of enzymes driving this metabolic pathway 35,36. We found that
the addition of nitrate significantly promoted WT growth under anaerobic conditions. However,
this growth-promoting effect was markedly inhibited by co-supplementation with tungstate
(Supplementary Fig. 7C). Notably, in the presence of both nitrate and tungstate, the addition of
increasing concentrations of molybdate progressively restored the anaerobic growth of the WT
(Supplementary Fig. 7C). In contrast, supplementation with nitrate, tungstate or molybdate had
no significant effect on the growth of ΔnarGHJI under anaerobic conditions (Supplementary Fig.
7D). Additionally, the growth of C. sakazakii WT and ΔnarGHJI showed no significant
difference in the presence of tungstate or molybdate under aerobic conditions (Supplementary
Fig. 7E). These results indicate that supplementation with tungstate disrupts nitrate respiration of
C. sakazakii, thereby inhibiting the bacterial growth under anaerobic conditions.

To access whether tungstate treatment would inhibit the infection of C. sakazakii, neonatal
rats were orally infected with C. sakazakii and then treated with tungstate. The results revealed
that tungstate treatment significantly reduced C. sakazakii burdens in the small intestine, colon,
blood, liver, and spleen at 48 hpi compared to untreated rats (Fig. 7D). In contrast, tungstate
treatment had no effect on the virulence of narGHJI in vivo (Fig. 7E). Meanwhile, we also
performed 16S rRNA sequencing on bacterial DNA from the caecal content of tungstate-treated
and control rats. The Chao index, Shannon index, and the PCoA based on Bray-Curtis distances
showed that tungstate treatment did not significantly alter intestinal bacterial diversity
(Supplementary Fig. 7F-G). Moreover, we assessed the relative abundance of the gut microbiota
in each group. Consistently, variations at the genus level also showed that tungstate treatment did
not alter the overall community structure (Supplementary Fig. 7H-I). The results indicate that
tungstate effectively inhibits C. sakazakii colonization in the GIT and its systemic dissemination
within the host, without significantly altering the gut microbiota, highlighting targeting nitrate
respiration may serve as a promising therapeutic intervention for C. sakazakii infections.

Methods
Ethics statement
All animal experiments were conducted in compliance with the guidelines outlined in the Guide
for the Care and Use of Laboratory Animals. All animal studies were conducted according to
protocols approved by the Institutional Animal Care Committee of Nankai University (Tianjin,
China) and were performed under protocol no. IACUC 2020030501.

Bacterial strains, primers, plasmids and growth conditions
Bacterial strains, plasmids and primers used in this study are provided in Supplementary Table 1
and 2. C. sakazakii strain ATCC 29544 spontaneously developed resistance to nalidixic acid was
used as the wild-type (WT) strain throughout this study. Mutant strains of C. sakazakii were
generated using the -red recombination system by pKD3 or pKD4 plasmid with
chloramphenicol or kanamycin resistance genes 58. A helper plasmid named pCP20 encoding the
FLP recombinases was used to eliminate the antibiotic resistance gene, when required.
Complementary plasmids were constructed using pACYC184, and electroporated into
corresponding mutant strains. For purification of ArcA-6×His, arcA was fused with pET28a
expression vector and transferred into E. coli BL21 (DE3). To generate the frr-lux reporter
fusion, the amplification products of frr promoter region was digested and cloned into the Xho I–
BamH I site of the plasmid pMS402, which carries a promoterless luxCDABE reporter gene
cluster. For ChIP DNA purification, arcA was tagged with 3×Flag, and fused with pTRC99A and
electroporated into C. sakazakii ATCC 29544. All the strains constructed in the study were
verified by sequencing. Bacteria were inoculated into fresh Luria-Bertani (LB) or M9 minimal
medium (1×M9 salts, 2 mM MgSO4, 0.1 mM CaCl2, 0.4% glucose) and grown at 37 ºC under
aerobic (250 rpm) or anaerobic (90 % N2, 5 % CO2, 5 % H2; Sheldon Manufacturing) conditions.
When needed, isopropyl β-D-thiogalactoside (IPTG) and antibiotics were added to the medium
at the following final concentrations: 0.1 mM IPTG, 50 μg/ml nalidixic acid, 25 μg/ml
chloramphenicol (Cm), 50 μg/ml kanamycin (Kan), and 10 μg/ml tetracycline (TE).

Northern blot analysis
Total RNA (5-10 µg) was normalized and samples were mixed with 2 × RNA loading buffer
(Beyotime, China). RNA was separated on 6% acrylamide gels containing 8 M urea after heating
65 ºC for 15 min. Bands were transferred to nylon membranes and crosslinked by UV.
Biotinylated oligonucleotides were used as probes and hybridizations performed in the
BeyoHyb™ Quick Hybridization Solution (Beyotime, China) at 42 ºC overnight. The
membranes were washed twice with low stringency wash solution at room temperature for 5 min
each time, and washed once at 42 ºC for 2 min. The subsequences were subjected to the Biotin
Northern Blot Kit (for Small RNA) Kit (Beyotime, China). Northern blots were visualized using
Amersham Imager 600, 5S ribosomal RNA (rRNA) was used as an internal control.

Race assays
Simultaneous determination of 5′ and 3′ ends of sRNA was performed using rapid amplification
of complementary DNA ends (RACE) kits (Invitrogen, USA) essentially as manufacturer’s
instructions. The oligonucleotides used for RACE are listed in Supplementary Table 2. In each
case, the generated PCR products by specific reverse primer of correct size were directly cloned
into the pEASY-T1 vector (TransGen, China). Plasmids with sRNA RACE inserts were
sequenced using M13 forward and reverse oligonucleotides.

Cell culture and C. sakazakii infection of macrophages
Murine macrophage cell line RAW264.7 (ATCC TIB-71) was obtained from the Shanghai
Institute of Biochemistry and cultured in RPMI 1640 supplemented with 10% (v/v) FBS
(Hyclone, USA) at 37 °C under a 5% CO2 atmosphere.
RAW264.7 cells were infected with WT or the indicated mutant or complemented strains that
grown to the logarithmic phase at a multiplicity of infection (MOI) of 100. The cell monolayers
were incubated for 45 minutes following infection, rinsed three times in PBS, and incubated for
another 1 hour in RPMI 1640 media containing gentamicin (100 μg/ml) to kill the extracellular
bacteria. Then each well containing bacterial cells were rinsed three times in PBS and
replenished with RPMI 1640 containing gentamicin (10 μg/ml) supplemented with 1 mM NaNO3
when required and incubated at 37 ºC with 5% CO2. From this point, the hour post-infection
(hpi) was deemed as 0. At 1, 4 and 24 hpi, the infected cells were washed three times with PBS
and lysed with 0.05% Triton X-100 (Solarbio, China). The intracellular bacteria were enumerated
on LB agar plates for CFU analysis or collected for qRT-PCR analysis. The fold intracellular
replication of the indicated strains was expressed as the CFUs recovered at 4 or 24 hpi relative to
those at 1 hpi.

RNA isolation and Quantitative RT–PCR (qRT–PCR)
Overnight culture of C. sakazakii WT strain and mutant strains were 1:100 subcultured in 20 ml
fresh LB medium at 37 ºC with shaking at 180 rpm until the exponential growth phase was
reached (OD600 = 0.6-0.8). For C. sakazakii collected from the cells, RAW264.7 cells were
infected by C. sakazakii WT or mutant strains at a MOI of 100:1 and collected at 1, 4 and 24 hpi.
For C. sakazakii collected from the small intestine and colon, 2- to 5-day-old Sprague-Dawley
rat pups were orally infected by 1 × 108 CFU of C. sakazakii WT or mutant strains, and collected
following the contents were removed at 48 hpi. For C. sakazakii treated with rifampicin, C.
sakazakii WT strain and mutant strains were incubated with 100 μg/ml of rifampicin and
collected at 0, 2, 4, 8, 16, 32 min. For the effect of oxygen levels on C. sakazakii, mid-
logarithmic phase C. sakazakii WT or mutant strains incubated in LB medium were collected and
reincubated in M9 minimal medium supplemented under both aerobic and anaerobic conditions
for 30 minutes.
Total RNA was extracted and purified using TRIzol reagent (Invitrogen, USA). RNA was treated
with DNase I at 37℃ for 30 min and successful removal of DNA contamination was verified by
PCR. Then the RNA was quantified and qualified by measuring the A260/A280 absorbance ratio
using a NanoDrop spectrophotometer (Thermo Fisher, USA).
cDNA synthesis was performed by StarScript III RT MasterMix (GenStar, China). qRT-PCR was
performed using 2×RealStar TPower SYBR qPCR Mix (Low ROX) (GenStar, China) on
QuantStudio 5 (Applied Biosystems, USA). The expression levels of C. sakazakii transcripts
were normalized to that of the rpoA gene 59, whereas those of the small intestine and colon
transcripts were normalized to GAPDH expression. The fold change in the expression of each
target gene was estimated using the 2−ΔΔCt method.

RNA sequencing
Overnight-cultured bacteria of C. sakazakii WT and CsrN strains were inoculated into fresh LB
medium with a ratio of 1:100 further cultured till the OD600 value reached approximately 0.6, and
then collected by centrifugation for RNA extraction with three biological replicates per sample.
The RNA quality was assessed with an Agilent 2100 Bioanalyzer (Santa Clara, CA) and agarose
gel electrophoresis (AGE). Illumina library preparation, sequencing, and data analysis were
conducted by Novogene (Bejing, China). Sequenced reads were checked for base quality,
trimmed where 50% of the bases were below quality score 20, and filtered to exclude adapters.
The clean reads were mapped to the C. sakazakii strain ATCC 29544 reference genome
(CP011047.1) by using the short-sequence alignment software Bowtie 2. Gene expression levels
were normalized and reported as FPKM. For differentially expressed genes (DEGs) analysis, raw
read counts were normalized by TMM method and DEGs was performed using the edgeR
package with a significance threshold of |log2(fold change)| > 1 and false discovery rate (FDR)-
adjusted p-values < 0.005 (Benjamini-Hochberg correction).

Growth assays
For aerobic growth, overnight cultures were diluted 1:1,000 into fresh LB, RPMI 1640 medium
or M9 minimal medium. When indicated, the media were supplemented with 40 mM nitrate, 0.5
mM tungstate, or 1 mM molybdate. 200 l aliquot was added to a 96 well microplate and
incubated at 37 °C at 180 rpm for 24 h aerobically. The optical density at 600 nm was measured
every 20 min. The experiment was performed in triplicate for each strain. For anaerobic growth,
cultures under exponential growth phase (OD600 = 0.6) at a final concentration of 1 × 108
CFU/ml, supplemented as follows: (1) 40 mM NaNO₃; (2) 0.5 mM tungstate; (3) 1 mM
molybdate; (4) 40 mM NaNO₃ and 0.5 mM tungstate; and (5) 40 mM NaNO₃ with both tungstate
and molybdate at the following molar ratios, 1:2, 10:1, and 100:1, and incubated under anaerobic
(90 % N2, 5 % CO2, 5 % H2; Sheldon Manufacturing) 60 growth conditions at 37 ºC. The bacteria
suspension was diluted with PBS and spread on agar plates for enumeration of bacterial CFUs at
1, 4 and 24 hours, respectively.

Protein expression, purification and electrophoretic mobility shift assays (EMSAs)
The constructed pET28a-ArcA plasmid was transformed into E. coli BL21 (DE3). pET-ArcA-
containing strain was cultured overnight in LB and 1:100 subcultured in fresh LB to OD600 = 0.6-
0.8 at 37 °C. Then IPTG was added to induce the expression of ArcA and growth at 16 °C for 20
h. The bacteria were lysed by using sonication and centrifuged at 4 °C (12,000 × g, 30 min). The
supernatant containing ArcA was subsequently purified using HiTrap Ni2+-chelating column.
Protein concentration was determined according to the Bradford method 61.
CsrN fragments of the promotor regions and point mutant were amplified by PCR and purified
using StarPrep DNA Gel Extraction Kit (GenStar, China), as well as the DNA fragments of kana
DNA (negative control). The DNA fragment (40 ng) was incubated at 37 °C for 40 min with
different concentrations of 6× His-tagged ArcA protein (0-1.75 M) in 20 l binding buffer (100
mM Tris-HCl [pH 7.4], 100 mM KCl, 10mM MgCl, 10% glycerol, and 2 mM dithiothreitol).
When needed, 30 mM acetyl phosphate was added to the binding system. Samples were then
separated on 6% polyacrylamide gel in 0.5 × Tris-Borate-EDTA (TBE) for electrophoresis. DNA
fragments were stained with GelRed (Life-iLab, China).

mRNA stability assay
Overnight culture of C. sakazakii WT strain and mutant strains were 1:100 subcultured in 20 ml
fresh LB medium at 37 ºC with shaking at 180 rpm until the exponential growth phase was
reached (OD600 = 0.6). The medium was then centrifuged and the bacteria were re-suspended
with M9, to which rifampicin (100 μg/ml) was added. At each indicated time point after
rifampicin treatment, the same volume of bacteria was collected, and the corresponding RNA
was extracted immediately for qRT-PCR.

REMSAs
The target DNA templates were transcribed into CsrN, CsrN+ (positive control), CsrNmut sRNA,
5′ UTR-narGHJI, 5′ UTR-narGHJImut mRNA using T7 High Efficiency Transcription Kit
(TransGen, China) and purified by MagicPure® RNA Beads (TransGen, China). REMSA was
performed with CsrN, CsrNmut sRNA (0, 10, 20 and 30 μM) and 5′ UTR-narGHJI, 5′ UTR-
narGHJImut mRNA or CsrN+ (10 μM) in REMSA binding buffer (10 mM HEPES [pH 7.3], 20
mM KCl, 2.4 mM MgCl2, and 2.4 mM DTT). The reaction mixtures were incubated for 3 min at
85 ºC and then at 37 ºC for 45 min. RNA was separated by 6% native polyacrylamide gel
electrophoresis (PAGE) using a Native-PAGE Preparation kit (Sangon, China) and stained with
GelRed (Life-iLab, China) for 10 min, and imaged using a gel imaging system (Tanon).

DNA affinity pull-down assay
DNA pull-down assays were conducted as previously described 62, with modifications to
optimize protein-DNA interaction capture. A biotin-labeled DNA fragment encompassing the
CsrN promoter region was PCR-amplified from C. sakazakii and immobilized on streptavidin-
coated Dynabeads (Invitrogen, USA). The DNA-bound beads were incubated with crude protein
extracts from C. sakazakii wild-type strains under optimized binding conditions. Following
rigorous washing to remove nonspecifically bound proteins, the retained proteins were eluted
using a high-salt buffer (500 mM NaCl), resolved by SDS-PAGE, and visualized through
Coomassie Brilliant Blue staining. Protein bands of interest were excised from the gel, subjected
to in-gel tryptic digestion, and analyzed by matrix-assisted laser desorption ionization-time-of-
flight tandem mass spectrometry (MALDI-TOF-MS/MS). Peptide sequences and mass spectra
were compared against the NCBI non-redundant database for protein identification.

Animal model
2- to 5-day-old Sprague-Dawley rats were purchased from Beijing Vital River Laboratory
Animal Technology Co., Ltd. (licensed by Charles River), and housed in specific-pathogen-free
(SPF) mouse facilities. For colonization experiments, rats were infected orally with 108 CFU of
C. sakazakii WT strain and mutant strains in the logarithmic phase of growth 63. 48 hours later,
blood specimens were collected for bacterial cultures (CFU). Small intestine and colon were
collected and the contents were removed. Liver, spleen small intestine and colon were weighed
and homogenized with PBS, and spread on agar plates for enumeration of bacterial CFUs. For
survival assays, 2- to 5-day-old Sprague-Dawley rat pups were infected orally with 107 CFU of
C. sakazakii WT strain and mutant strains in the logarithmic phase of growth 64. The numbers of
animals alive was recorded every 8 hours.

In vivo bioluminescence imaging (BLI)
The DNA fragment of frr promotor were amplified, purified, and digested with BamH I/Xho I.
The products were ligated to pMS402 plasmid to construct of lux fusion plasmids 23, which was
introduced into C. sakazakii WT, CsrN, narGHJI and CsrNnarGHJI strains, respectively.
The plasmids and insertions were verified by Sanger sequencing. 2- to 5-day-old Sprague-
Dawley rats were infected by oral gavage with the strains harboring lux fusions (108 CFU). At 48
hours, rats were imaged and followed by in vivo BLI using the in vivo imaging system Lumina II
(Caliper Life Sciences, USA). Data acquisition and analysis were performed using living image
software (Caliper Life Sciences, USA).

Lux reporter assay
For lux reporter fusion, the 5′ UTR or 5′ UTRmut of narGHJI were cloned into the pMS402
plasmid through BamH I/Xho I, respectively. For sRNA overexpression plasmids, the DNA
fragments of CsrN and CsrNmut were cloned into the pNM12 plasmid through Msc I/EcoR I,
respectively. The empty pNM12 vector was labeled as control. The lux reporters and sRNA
overexpression plasmids were then transformed into CsrN strain. The plasmids and insertions
were verified by Sanger sequencing. The expression of lux-based reporters from CsrN strains
growing in liquid culture with 0.1% arabinose (inducing the expression of sRNA) was measured
using the Spark multimode microplate reader (Tecan, Switzerland).

Measurement of nitrate and nitrite concentration
The small intestine and colon of normal rat pups or that infected with C. sakazakii WT were
collected, removed the contents and homogenized in PBS containing protease inhibitors of
choice, then centrifuge at 10,000 × g for 20 minutes at 4 ºC. Transfer the supernatant to another
tube as tissue samples. The cell supernatant of RAW264.7 infected with C. sakazakii WT at 1, 4
and 24 hpi, respectively, and uninfected cell supernatant were collected as cell samples. The
following procedures were carried out according to the specification of Nitrate/Nitrite
Colorimetric Assay Kit (Cayman, USA). Firstly, to determination concentration of total nitrate
and nitrite, 80 l samples were added to 96-wells, then 10 l nitrate reductase cofactors
preparation and 10 l nitrate reductase enzyme preparation was added. After incubation for 2-3
hours, 50 µl of Griess reagent 1 and Griess reagent 2 was added, and absorbance was measured
at 540nm about 10 minutes later. Secondly, to determination concentration of nitrite, 100 l
samples were added to 96-wells, then 50 µl of Griess reagent 1 and Griess reagent 2 was added,
and absorbance was measured at 540nm about 10 minutes later. The nitrate concentration is
equal to the total nitrate and nitrite concentration calculated using the nitrate standard curve
minus the nitrite concentration calculated using the nitrite standard curve.

Cytotoxicity Assay
A lactate dehydrogenase (LDH) release assay was performed to assess cytotoxicity during
bacterial infection. Following the infection of RAW264.7 cells with C. sakazakii WT, the
indicated mutant or complemented strain, culture supernatants were collected at 1, 4 and 24 hpi.
The supernatants from uninfected cells were used as a negative control. Cytotoxicity was
quantified using the Cytotox 96 non-radioactive cytotoxicity assay (Promega), and the
percentage of cytotoxicity was calculated according to the manufacturer’s instructions.

16S rRNA gene sequencing and analysis
Caecal content samples were collected from 2- to 5-day-old rat pups, which were allocated into
an untreated control group (Mock) and a tungstate-treated group (Tungstate; 48 hours of
treatment), with 8 animals per group, and extracted the total DNA. The V3-V4 hypervariable
region of the bacterial 16S rRNA gene was amplified by PCR using the primer pair 338F (5’-
ACTCCTACGGGAGGCAGCA-3’) and 806R (5’-GGACTACHVGGGTWTCTAAT-3’). The
resulting PCR products were purified, and sequencing libraries were constructed. Paired-end
sequencing was subsequently performed on a NextSeq 2000 system (Illumina, USA). All the
analysis was carried out on the Majorbio platform on the website at https://cloud.majorbio.com.
α-diversity were calculated by software of Mothur (http://www.mothur.org/wiki/Calculators,
version v1.30.2), and Principal Co-ordinates Analysis (PCoA) were conducted for the analysis of
β-diversity. The comparisons among the microbiota compositions on the genus level were
conducted using Vegan v2.5-3 package.

Statistics
For each experiment, the number of independent replicates is indicated in the figure legends,
along with the corresponding statistical test used. Statistical significance was analyzed with
GraphPad Prism 9.5.0 software (GraphPad Inc). Data were analyzed with either a Student’s t-test
to compare two conditions with normal distributions or a Mann–Whitney U-test for two
conditions without normal distributions. For assessing statistical significance among three or
more groups, a one-way analysis of variance (ANOVA), two-way ANOVA or Dunnett’s test were
utilized as indicated. Survival analysis was used log-rank (Mantel-Cox) test. Differences were
considered significant at p < 0.05.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The authors describe the characterization of a novel sRNA that regulates the nar operon and allows C. sakazakii to metabolize nitrate under anaerobic conditions. Moreover, they provide evidence that tungsten supplementation can suppress C. sakazakii infection in an animal model, as has been previously shown for Enterobacteriaceae sp. C. sakazakii has been implicated in infections of infants, often associated with contaminated formula. This is a timely report with a logical flow of experiments from initial regulator discovery to functional consequence to validation in an animal model. However, there are numerous issues that are currently unclear in the current manuscript as detailed below:\n1. The RNA sequencing section is woefully incomplete. There is no details of how many samples were sequenced, their growth conditions, how the sequencing was done quality controlled or mapped to the reference strain. Were the authors able to confirm deletion of the csrN transcript in their RNA seq dataset and did the deletion have a polar effect on the adjacent yhoB & degQ genes?\n2. The authors report using FPKM as input into edgeR for differential abundance analysis. This is incorrect, as edgeR’s TMM normalization method assumes that raw counts are input. As such, this analysis must be redone to properly assess differential abundance. Are the author reporting uncorrected or corrected p values (line 557)?\n3. For the bacterial growth assay, only aerobic conditions are mentioned (line 561). Given the presentation of both aerobic/anaerobic growth assays, can the authors clarify this?\n4. For the bacterial replication assay, the authors note that the cultures were shaken under aerobic conditions (line 568-9), but do not make this comment for the anaerobic conditions. Were the cultures grown statically under anaerobic conditions and would that impact the results?\n5. The authors present growth data as both classical cell density measurements (eg. S5C S7AB) and also as log2(relative replication ratio). How is relative replication ratio calculated and why is this used instead of growth curves in the main manuscript but not the supplemental?\n6. The use of nitrate by C. sakazakii appears to primarily occur under anaerobic conditions. The authors employ both aerobic and anaerobic culturing at various points in the manuscript and do not always clearly state or indicate on figures whether the growth was done aerobically/anaerobically (e.g. FigS2, Fig5E, Fig 6ACJK and others). This should be corrected.\n7. What other transcripts would be predicted to be regulated by csrN in the same manner as narGHIJ based on binding to the 5’UTR?\n8. The authors tested mutants in ΔnarGHJI, and ΔnarK for nitrate respiration, but didn’t appear to test the ΔcsrN mutant. Is the ΔcsnN mutant also impacted in nitrate respiration?\n9. The authors tested the ΔcsnNΔnarGHJI in vivo, but do not appear to have tested the ΔcsnN mutant. Is the ΔcsrN mutant also impacted in vivo?\n10. Related to above comments, the authors show that the CsrNmut is impacted in colonization. What validation experiments did the authors do to determine whether transcripts other than narGHIJ would be impacted by this mutation? Did the authors test a strain where both the csrN and narGHIJ 5’UTR were mutated to see if this restores function?\n11. The authors suggest that tungsten supplementation in vivo impacts infection through nitrate metabolism as the WT strain showed reduced colonization whereas the ΔnarGHJI mutant appeared unaffected. The authors should demonstrate that tungsten does not impact in vitro growth of the WT & ΔnarGHJI strains (aerobically/aerobically) and that it also impacts nitrate utilization in the WT strain. Titrating with molybdenum (the presumed cofactor that tungsten is replacing) would also be a very useful mechanistic demonstration.\n12. The model presented in figure 8 has several aspects that should be improved:\na. It seems to imply that C. sakazakii is a gram positive (single membrane layer between extracellular /cytoplasm space)\nb. What is “inflammatory factor” in this model?\nc. Why have nitrate represented by both a green dot and also write out NO3-?\nd. The model should include the proposed mechanism whereby tungsten would inhibit this pathway\ne. Explicitly incorporating host elements (e.g NOS via Nos2) into the model and its interaction with C. sakazakii would be useful.\n13. Did the authors assess the impact of tungsten on their rat microbiomes? While previous report suggested that tungsten may have minimal impact on mouse microbiotas, this should be validated in this rat model.\n14. On a similar line of thought, the authors tone down the statement that tungsten does not adversely affect the commensal microbiota (line 458), as a single paper is being referenced. This is likely an oversimplification as numerous bacteria employ molybdate cofactors for and would be susceptible to the same tungsten-mediated poisoning. Moreover, any therapies for targeting C. sakazakii would naturally be employed in infants who may be more susceptible to microbiota alterations given their young age.\n15. Figure 5B shows an increase in nitrate production in RAW264.7 cell over time. Did the authors do a mock infection to determine if nitrate is being induced by infection vs accumulating over time?\n16. The statement in lines 428-430 stating that “aerobic respiration” being dispensable for virulence is to broad. Only single deletion mutants were tested and there could be compensation between the pathways both in vivo and in vitro should the appropriate substrates be present.", "reviewer_2": "The authors reported a signal transduction pathway centered on a novel sRNA, CsrN, which facilitates C. sakazakii in utilizing nitrate respiration in response to oxygen-limited environments within the host, thereby enhancing its virulence in vivo. There were some major concerns need to be addressed.\n1. Lines 21 and 54, C. sakazakii infections are usually associated with powdered infant formula, but the link with rice-based supplementary food remains contentious. The authors should provide sufficient references—specifically clinical case reports with supporting epidemiological data—to substantiate this assertion regarding rice-based products.\n2. Line 145-150, It remains unclear whether the reported bacterial counts represent C. sakazakii exclusively or include other microorganisms. Please specify the selective/differential medium used for plating, as this critically impacts data interpretation. Figure 2C-D presents survival rates and bioluminescent imaging for wild-type and ΔCsrN. To establish genetic causality, data for the complemented strain (ΔCsrN+pCsrN) must be included. This control is essential to demonstrate that observed phenotypes result specifically from CsrN deletion rather than secondary mutations.\n3. Beyond the CGG motif in hairpin loop of CsrN, whether other nucleotides within the complementary region between 5′ UTR of narGHJI mRNA and the CsrN sRNA affect their binding interaction?\n4. To establish that CsrN increases the expression of narGHJI by influencing its mRNA stability, the authors should perform genetic complementation experiments using a CsrN-expressing plasmid in ΔCsrN strains.\n5. Line 231-234, the survival of ΔCsrNΔnarGHJI exhibited no significant difference from that of ΔnarGHJI, the author speculated CsrN deletion did not affect the bacterial survival in macrophages in a ΔnarGHJI background. This experiment lacked the essential control group: the ΔCsrNΔnarGHJI strain complemented with CsrN (ΔCsrNΔnarGHJI + pCsrN).\n6. The most significant limitation of this study is that, beyond comparing bacterial loads, it lacks supporting pathological evidence to substantiate the changes in C. sakazakii virulence following gene knockout. Crucially, data demonstrating differences in key indicators of pathogenicity – such as cytopathy, cytotoxicity/cell damage, inflammatory cytokine levels, and histopathology of affected organs – are missing. Furthermore, the ultimate cause of death in the infected mice remains undefined.\nWhile the ΔnarGHJI mutant group showed approximately 20% survival at 96 hours, all mice infected with the wild-type (WT) strain and the other knockout mutants (including the ΔCsrNΔnarGHJI double mutant) succumbed to infection. The observed difference in time-to-death between WT-infected mice and knockout mutant-infected mice was only about 24 hours. This relatively short time difference raises questions about the magnitude and biological significance of the attenuation in virulence attributed to the gene knockouts.\nConsequently, while the study effectively demonstrates that CsrN and narGHJI knockouts impact the total bacterial load in vivo, it lacks direct evidence linking these genetic modifications to a definitive change in C. sakazakii virulence.", "reviewer_3": "This manuscript investigates a regulatory pathway in Cronobacter sakazakii that connects the global regulator ArcA, the small RNA CsrN, and the nitrate reductase operon narGHJI. RNA-seq identifies narGHJI as a CsrN-upregulated operon. The authors demonstrate that CsrN base pairs directly with the narG 5′UTR to stabilize its mRNA, and that ArcA binds to the csrN promoter to activate transcription. Evidence is provided from different models linking this regulatory cascade to survival and pathogenesis. Finally, they show that sodium tungstate, a nitrate reductase inhibitor, reduces bacterial burden and prolongs host survival. A noteworthy strength is that the phenotypes are clearly observed in a ΔcsrN strain. For many sRNAs, only overexpression reveals an effect, but here the loss-of-function phenotype is strong and reproducible. This underscores the biological importance of CsrN and strengthens the conclusions.\nOverall, the study is conceptually appealing: it defines a regulatory chain from a global transcriptional regulator (ArcA), through an sRNA (CsrN), to a metabolic operon (narGHJI), establishing a direct link between regulatory RNA biology, metabolism, and virulence. Nevertheless, several comments and questions, listed below, could further improve the manuscript.\n1. Persistence and infection strategy\nThe data clearly support bacterial persistence in host niches—specifically survival in macrophages and colonization of inflamed, oxygen-limited tissues—driven by nitrate respiration. The manuscript uses “persistence” in this ecological/pathophysiological sense. However, antibiotic persistence (tolerance to bactericidal drugs) is not addressed, even though nitrosative stress is known to shape persistence in enteric pathogens. Incorporating antibiotic-challenge assays in infected cells would broaden the impact and connect the findings to the antibiotic persistence field.\n2. ΔarcA virulence phenotype\nThe ΔarcA strain is analyzed in depth for gene expression: ArcA binds the csrN promoter and is required for csrN and narGHJI induction under anaerobic conditions and during infection. While this firmly establishes transcriptional control, I did not see direct in vivo virulence data for ΔarcA (e.g., reduced colonization or systemic spread in the rat model). Including ΔarcA infection data with complementation would fully close the regulatory loop from sensor (ArcA) to virulence phenotype.\n3. Role of Hfq\nSince most sRNAs in enterobacteria require Hfq for stability and function, the role of Hfq in C. sakazakii deserves clarification. Does Hfq bind and stabilize CsrN as in other Enterobacteriaceae? If this has not been directly tested, a brief discussion of this point would help readers interpret the generalizability of the mechanism.\n4. Conservation of CsrN\nThe manuscript shows that CsrN is conserved across C. sakazakii species. What about conservation in other Gram-negative bacteria? If CsrN or a homolog exists more broadly, that could expand the significance of the findings.\n5. Pathogen lifestyle and infection model\nIs C. sakazakii considered a true intracellular pathogen? Are macrophages its preferred niche in vivo, or is the intracellular stage primarily a transient step during dissemination? The transition in the text to macrophage assays is quite abrupt. A fuller justification of the macrophage model’s relevance would help contextualize the intracellular findings.\n6. Choice of animal model\nThe rationale for using neonatal rats instead of mice is not fully explained. Is this a validated or preferential model for C. sakazakii infection? A short justification would help. In addition, the term “systemic infection” may be too strong for a gavage model where blood samples were not collected to quantify bacteremia. It would be clearer to describe dissemination based on colonization of distal organs."}, "author_response": "Reviewer #1 (Remarks to the Author):\nThe authors describe the characterization of a novel sRNA that regulates the nar operon\nand allows C. sakazakii to metabolize nitrate under anaerobic conditions. Moreover,\nthey provide evidence that tungsten supplementation can suppress C. sakazakii\ninfection in an animal model, as has been previously shown for Enterobacteriaceae sp.\nC. sakazakii has been implicated in infections of infants, often associated with\ncontaminated formula. This is a timely report with a logical flow of experiments from\ninitial regulator discovery to functional consequence to validation in an animal model.\nHowever, there are numerous issues that are currently unclear in the current manuscript\nas detailed below:\n1. The RNA sequencing section is woefully incomplete. There is no details of how many\nsamples were sequenced, their growth conditions, how the sequencing was done quality\ncontrolled or mapped to the reference strain. Were the authors able to confirm deletion\nof the csrN transcript in their RNA seq dataset and did the deletion have a polar effect\non the adjacent yhoB & degQ genes?\nResponse: Thank you for your valuable suggestions. The details in method of RNA-\nsequencing were added in lines 621-636 of the revised manuscript.\nTo validate the knockout of CsrN, the RNA sequencing reads were aligned to the\nreference genome (CP011047.1) using BWA-MEM (v0.7.19). The ΔCsrN strain\nexhibited a complete absence of read coverage across the 5′ region of CsrN, whereas\nthe wild-type strain exhibited uniform transcriptional coverage, confirming the\nsuccessful disruption of CsrN expression.\nIn our RNA-seq result, we found that degQ and yhcB were down-regulated in ΔCsrN\n(12.5- and 3.1-fold, respectively). The reduced expression of degQ was due to the\ndeletion of its promoter region in ΔCsrN, which overlaps with CsrN. We infected\nRAW264.7 with WT or ΔdegQ (mutated the start codon in the chromosome, from ATG\nto CTG), and no significant difference in intracellular survival was observed between\nthese two strains (Response Fig. 1a). Moreover, we investigated whether degQ\ninfluences bacterial virulence in vivo by orally infecting neonatal rats with WT or\nΔdegQ. The bacterial burdens in the small intestine, colon, blood, liver, and spleen\nshowed no significant differences between the two groups (Response Fig. 1b). These\ndata indicate that the deletion of degQ does not influence bacterial virulence.\nAn 8-nucleotide complementary region between the CsrN sRNA (UCAUGAAC) and\nthe yhcB mRNA (GUUCAUGA) were predicted using IntaRNA, indicating that CsrN\nmay directly regulate the expression of yhcB. Previous studies have shown that deletion\nof yhcB affects bacterial growth by compromising cell envelope integrity in\nEscherichia coli (PLoS Genet. 2021;17(12):e1009586). Thus, to investigate whether\nthe influence of CsrN on bacterial virulence is mediated via yhcB, we specifically\ndisrupted this predicted regulatory interaction. Within the predicted base-pairing region,\nfour-nucleotides in CsrN (underlined) lie within a hairpin loop and are complementary\nto the yhcB mRNA (underlined, which includes the initiation codon of yhcB), and may\nplay a key role in the interaction. To abolish this interaction, we generated a CsrNmut-y\nstrain carrying a mutation in the CsrN binding site (UCAU to ACUU). qRT-PCR assays\nshowed that the yhcB expression in CsrNmut-y exhibited no significant difference from\nthat of ΔCsrN strains (Response Fig. 1c), but was significantly decreased compared to\nthat of WT strain (Response Fig. 1c), suggesting that the mutation in CsrN successfully\nabolished the CsrN-mediated regulation on yhcB. In contrast, the expression of\nnarGHJI in CsrNmut-y and WT strains exhibited no significant difference (Response Fig.\n1c). The decreased yhcB expression in the CsrNmut-y strain enabled us to assess the\nimpact of the CsrN-yhcB regulatory interaction on virulence. Infection of RAW264.7\nwith WT or CsrNmut-y showed that the survial of CsrNmut-y in macrophages exhibited no\nsignificant difference from that of WT strain (Response Fig. 1a). Moreover, we also\nshowed that there was no significant difference in bacterial burdens in small intestine,\ncolon, blood, liver and spleen between rats infected with CsrNmut-y or WT strain\n(Response Fig. 1b). These data indicate that the CsrN-yhcB regulatory interaction does\nnot influence bacterial virulence.\nCollectivelly, these data showed that the impaired survival in macrophages and\ndissemination in the host observed in ΔCsrN are not due to the downregulation of degQ\nor yhcB.\nResponse Fig. 1 (a) Bacterial burdens of C. sakazakii WT, ∆degQ or CsrNmut-y strain in\nRAW264.7 at 1, 4 and 24 hpi. n = 3 independent experiments. (b) Bacterial counts recovered from\nthe blood, liver, spleen, small intestine, and colon of 2- to 5-day-old rats orally infected with 1 ×\nPCR analysis of yhcB and narGHJI expression in the C. sakazakii WT, ∆CsrN and CsrNmut-y\nstrains in LB medium. n = 3 independent experiments.\n2. The authors report using FPKM as input into edgeR for differential abundance\nanalysis. This is incorrect, as edgeR’s TMM normalization method assumes that raw\ncounts are input. As such, this analysis must be redone to properly assess differential\nabundance. Are the author reporting uncorrected or corrected p values (line 557)?\nResponse: We appreciate the reviewer’s insightful comment. We apologize for the\ninaccurate description in the Methods section. In our analysis, we did not use FPKM\nvalues as input for edgeR; instead, we used raw read counts to generate the DGEList\nobject and performed TMM normalization as required by edgeR. In addition, the p-\nvalues reported in the differential abundance analysis were FDR-corrected using the\nBenjamini-Hochberg method. We’ve revised this part in Methods section (lines 628-\n636) accordingly to correctly reflect the use of raw counts and the reporting of FDR-\nadjusted p-values.\n3. For the bacterial growth assay, only aerobic conditions are mentioned (line 561).\nGiven the presentation of both aerobic/anaerobic growth assays, can the authors clarify\nthis?\nResponse: Thank you for the comments. We apologize for the lack of clarity in our\noriginal manuscript, in which “Bacteria replication assay” (lines 564-571 of the original\nmanuscript) referred in fact to the method for anaerobic growth assays. We have now\ncombined these two part into a single section titled “Growth assays” and have revised\nthe content for better clarity and accuracy. The updated section can be found in lines\n637-651 of the revised manuscript.\n4. For the bacterial replication assay, the authors note that the cultures were shaken\nunder aerobic conditions (line 568-9), but do not make this comment for the anaerobic\nconditions. Were the cultures grown statically under anaerobic conditions and would\nthat impact the results?\nResponse: Thank you for the comments. You are correct that under standard anaerobic\nconditions, bacterial cultures were grown statically (without shaking). This approach is\nwidely used in anaerobic microbiology, as shaking is unnecessary to achieve or\nmaintain anaerobiosis. In contrast, shaking under aerobic conditions is essential to\nfacilitate adequate oxygen transfer for bacterial growth. This method of using static\ncultures for anaerobic growth is well-established in the literatures (e.g. Nat Commun.\n2023;14(1):642; Cell Host Microbe. 2025;33(8):1321-1332.e9; Cell Host Microbe.\n2017;22(3):291-301.e6). In addition, to directly investigate whether shaking would\nimpact our results, we cultured the WT strain under anaerobic condition with shaking,\nand the expression of CsrN and narGHJI in this condition was detected and compared\nwith that of WT strain grown under anaerobic condition statically. We found that there\nwas no significant difference in the expression of CsrN or narGHJI between these two\nconditions (Response Fig. 2a), suggesting that shaking did not impact the expression of\nCsrN and narGHJI under anaerobic condition. Moreover, we also determined the\ngrowth curves of WT and ΔnarGHJI under anaerobic condition with shaking. We found\nthat ΔnarGHJI exhibited reduced growth compared to WT, consistent with the result\nunder anaerobic condition statically (Response Fig. 2b, Fig. 5H). These results suggest\nthat the observed differences in gene expression (Fig. 6G) and bacterial replication (Fig.\n5H) under anaerobic conditions in our study are not attributable to artifacts introduced\nby the cultivation method.\nResponse Fig. 2 (a) qRT-PCR analysis of CsrN and narGHJI expression in the C. sakazakii WT\nunder anaerobic conditions grown statically or with shaking. (b) Replication of C. sakazakii WT\nand narGHJI strains in LB medium under anaerobic conditions grown with shaking. Relative\nreplication ratio was the fold change in growth at 1, 4, and 24 hours / 0 hours. n = 3 independent\nexperiments.\n5. The authors present growth data as both classical cell density measurements (eg. S5C\nS7AB) and also as log2(relative replication ratio). How is relative replication ratio\ncalculated and why is this used instead of growth curves in the main manuscript but not\nthe supplemental?\nResponse: Thank you for the comments. We apologize for the lack of clarity in our\ndescription. In our study, classical cell density measurements were used in growth\nassays under aerobic conditions (Supplementary Fig. 2A-B, 4A-B, 5C, 7A-B and 7E).\nHowever, due to the growth defect of ΔnarGHJI and ΔnarK under anaerobic conditions,\nbacterial growth at specific time points (e.g., 0, 1, 4, and 24 hours) was measured using\nplate counting, which offers higher sensitivity at low cell densities (Res Microbiol. 2025\nSep 20:104340; Cell Rep. 2022;38(1):110180). The 0-hour time point corresponds to\nthe moment when bacterial cultures reached OD = 0.6, at which the bacterial density\n600\nwas approximately 1×108 CFU/mL. This time point was used as the baseline to\ncalculate the fold change in bacterial growth at 1, 4, and 24 hours. By plotting the log2-\ntransformed fold change, we generated a normalized representation of replication over\ntime. This visualization offers a significant advantage over a standard bar graph of CFU,\nas it specifically highlights the rate and extent of bacterial replication from the onset of\nthe experiment (Fig. 5H). Consistently, the relative replication ratio was also used to\ndetermine the effect of nitrate on the survival of WT, ΔnarGHJI and ΔnarK in\nmacrophages (Fig. 5I). The relative replication ratio at 4 and 24 hpi were normalized to\nthe bacterial load at 1 hpi for each respective strain, as intracellular survival was\nmeasured at 1, 4, 24 hpi (Fig. 2A and 4A). The relevant calculation methods have been\nadded to the Figure legends in lines 1036-1040 and 1062-1063 of the revised\nmanuscript.\n6. The use of nitrate by C. sakazakii appears to primarily occur under anaerobic\nconditions. The authors employ both aerobic and anaerobic culturing at various points\nin the manuscript and do not always clearly state or indicate on figures whether the\ngrowth was done aerobically/anaerobically (e.g. FigS2, Fig5E, Fig 6ACJK and others).\nThis should be corrected.\nResponse: Thank you for the comments. We apologize for the lack of clarity in our\noriginal description. We have added the relevant information regarding the use of\naerobic or anaerobic culturing on Fig. 3D, 5E, 6A, 6C, and Supplementary Fig. 2A-B,\n4A-B, 5C-E, 6A, 6D, 6F-L and 7A-E.\nMoreover, we also performed additional experiments to detect the CsrN expression in\nWT and ΔarcA, and narGHJI expression in ΔCsrN and ΔCsrNΔarcA under aerobic\nconditions (Supplementary Fig. 6A and 6D). We found that in contrast to the\ndownregulation of CsrN in ΔarcA under anaerobic conditions (Fig. 6A), the CsrN\nexpression exhibited no significant difference between WT and ΔarcA strains under\naerobic conditions (Supplementary Fig. 6A). We also showed that the expression of\nnarGHJI exhibited no significant difference between ΔCsrN and ΔCsrNΔarcA under\naerobic conditions (Supplementary Fig. 6D). These data are consistent with the\nactivation of ArcA’s regulatory function under anaerobic conditions and have been\nadded in lines 363-366 and 370-373.\n7. What other transcripts would be predicted to be regulated by csrN in the same manner\nas narGHJI based on binding to the 5’UTR?\nResponse: Thank you for the comments. In our study, we first compared the\ntranscriptional profiles of WT and ΔCsrN using RNA-seq and found that 29 genes,\nincluding narGHJI were significantly down-regulated, while 95 genes were up-\nregulated in the absence of CsrN (Fig. 3A). We then employed IntaRNA, a\ncomputational tool for predicting RNA-RNA interactions, to assess whether CsrN base-\npairs with the 5′ UTR of narGHJI mRNA.\nTo further investigate whether CsrN regulates other transcripts through interactions\nwith their 5′ UTRs, we used IntaRNA to predict its potential base-pairing with other\n121 differentially expressed genes (excluding narH, narJ and narI). This analysis\nrevealed that only degQ exhibited a potential base-pairing interaction between its 5′\nUTR and the single-stranded RNA domain of CsrN stem-loop structure. In addition, we\nfound a four-nucleotide sequence within a CsrN hairpin loop that is complementary to\na region of yhcB mRNA (which includes its initiation codon near the 5′ UTR),\nindicating a possible regulatory role of CsrN on the expression of yhcB. However, as\nour response to your Comment 1, the regulation of CsrN on degQ and yhcB is not\nrelated with bacterial virulence.\n8. The authors tested mutants in ΔnarGHJI, and ΔnarK for nitrate respiration, but didn’t\nappear to test the ΔcsrN mutant. Is the ΔcsnN mutant also impacted in nitrate respiration?\nResponse: Thank you for the comments. To investigate whether nitrate respiration\ninfluences the growth of ΔCsrN, we performed additional bacterial growth experiments\nin LB medium under anaerobic conditions, with or without nitrate supplementation.\nThe results showed that WT strain exhibited slightly enhanced replication compared to\nΔCsrN at 1, 4, 24 hours without nitrate supplementation; however, the differences were\nnot statistically significant (Supplementary Fig. 5D). This result may be attributed to\nthe low concentration of nitrate present in LB medium, as no significant difference in\ngrowth between WT and ΔCsrN was observed in M9 medium, which lacks nitrate\n(Response Fig. 3). Moreover, nitrate supplementation promoted the anaerobic growth\nof both the WT and ΔCsrN strains. Notably, the growth of WT was significantly greater\nthan that of ΔCsrN under this condition (Supplementary Fig. 5D). Furthermore, we also\nperformed macrophages infection assays in WT and ΔCsrN strains with or without\nnitrate supplementation. We found that nitrate supplementation significantly enhanced\nthe intracellular survival of both WT and ΔCsrN strains, and WT also exhibited\nsignificantly greater survival than ΔCsrN under this condition (Supplementary Fig. 5E).\nThe reduced growth of ΔCsrN compared to WT under nitrate-supplemented conditions\nare probably due to its lower expression of narGHJI compared to the WT strain.\nThese results have been added in lines 341-352 of the revised manuscript.\nResponse Fig. 3 Replication of C. sakazakii WT, CsrN and narGHJI strains in M9 medium\nunder anaerobic conditions. Relative replication ratio was the fold change in growth at 1, 4, and 24\nhours / 0 hours. n = 3 independent experiments.\n9. The authors tested the ΔcsnNΔnarGHJI in vivo, but do not appear to have tested the\nΔcsnN mutant. Is the ΔcsrN mutant also impacted in vivo?\nResponse: Thank you for the comments. We have investigated the role of CsrN in\nbacterial pathogenicity in vivo by orally infecting 2- to 5-day-old rats with WT, CsrN\nand ΔCsrN complemented strains. The results showed that rats infected with CsrN\nexhibited significantly decreasd bacterial burdens in the small intestine, colon, blood,\nliver, and spleen compared to those infected with WT and ΔCsrN complemented strain,\nsuggesting that CsrN contributes to both GIT colonization and systemic dissemination\nby C. sakazakii within the host (Fig. 2B). Additionally, we have tested the survival\ncurves of rats orally infected with WT, CsrN or ΔCsrN complemented strains. The\nresult showed that rats infected with CsrN exhibited prolonged survival times\ncompared to those infected with WT or ΔCsrN complemented strains (Fig. 2C).\nBioluminescence imaging (BLI) showed that rats infected with CsrN-Lux exhibited\nsignificantly reduced systemic dissemination within the host compared with those\ninfected with WT-Lux (Fig. 2D). Moreover, we found that the levels of IL-6 and TNF-\nα, as well as the histopathology scores, were significantly higher in WT-infected rats\nthan those infected with CsrN (Fig. 2E-F and Supplementary Fig. 2D). These results\nsuggest that CsrN plays a critical role in enhancing the virulence of C. sakazakii in vivo.\n10. Related to above comments, the authors show that the CsrNmut is impacted in\ncolonization. What validation experiments did the authors do to determine whether\ntranscripts other than narGHJI would be impacted by this mutation? Did the authors\ntest a strain where both the csrN and narGHJI 5’UTR were mutated to see if this restores\nfunction?\nResponse: Thank you for your valuable suggestions. According to your suggestions,\nwe constructed a double mutant strain 5′ UTRmut-narCsrNmut by simultaneously mutating\nthe binding motif in 5′ UTR of narGHJI (CCG to GCU) and CsrN (CGG to AGC) on\nthe chromosome of WT. Then, we investigated whether this double mutation can restore\nbacterial survival in macrophages and systemic dissemination in the host, compared to\nthe single mutation in CsrN (CsrNmut) or 5′ UTR of narGHJI (5′ UTRmut-nar). We\nreperformed the infection assays using WT, CsrNmut, 5′ UTRmut-nar, and 5′ UTRmut-\nnarCsrNmut both in vitro and in vivo. The result showed that both 5′ UTRmut-nar and\nCsrNmut strains exhibited significantly reduced survival in macrophage at 1, 4, 24 hpi\ncompared to WT or 5′ UTRmut-narCsrNmut strain, whereas there was no significant\ndifference in survival between 5′ UTRmut-narCsrNmut and WT in macrophages\n(Supplementary Fig. 4H). Moreover, we also investigated whether double mutation\nrestored bacterial virulence in vivo. The results showed that 5′ UTRmut-nar and CsrNmut\nstrains exhibited markedly attenuated systemic dissemination in neonatal rats compared\nto WT or 5′ UTRmut-narCsrNmut strain, whereas the bacterial burdens in the small\nintestine, colon, blood, liver, and spleen of rats infected by 5′ UTRmut-narCsrNmut or WT\nexhibited no significant difference (Supplementary Fig. 4I). These results suggest that\nthe double mutation restores bacterial virulence compared to the single mutation in\nCsrN or narGHJI, confirming that the impaired survival of ΔCsrN in macrophages and\nits reduced systemic dissemination within host were due to the decreased expression of\nnarGHJI, rather than other transcripts. We have added these results in lines 291-295 of\nthe revised manuscript.\n11. The authors suggest that tungsten supplementation in vivo impacts infection through\nnitrate metabolism as the WT strain showed reduced colonization whereas the\nΔnarGHJI mutant appeared unaffected. The authors should demonstrate that tungsten\ndoes not impact in vitro growth of the WT & ΔnarGHJI strains (aerobically/aerobically)\nand that it also impacts nitrate utilization in the WT strain. Titrating with molybdenum\n(the presumed cofactor that tungsten is replacing) would also be a very useful\nmechanistic demonstration.\nResponse: We thank the reviewer for this crucial point. We have performed the\nfollowing experiments to address these points, and these data have been incorporated\ninto the revised manuscript (lines 438-449).\nNo difference in growth was observed between WT or ΔnarGHJI strain under aerobic\nconditions, regardless of the presence of tungstate or molybdate (Supplementary Fig.\n7E). The addition of nitrate significantly promoted WT growth under anaerobic\nconditions. However, this growth-promoting effect was markedly inhibited upon co-\nsupplementation with tungstate (Supplementary Fig. 7C). Notably, in the presence of\nboth nitrate and tungstate, the addition of increasing concentrations of molybdate\nprogressively restored anaerobic growth of the WT (Supplementary Fig. 7C). In\ncontrast, supplementation with nitrate, tungstate or molybdate had no significant effect\non the growth of ΔnarGHJI under anaerobic conditions (Supplementary Fig. 7D).\nThese results suggest that tungsten supplementation impacts the growth of C. sakazakii\nunder anaerobic conditions is medicated through its effect on bacterial nitrate\nmetabolism.\n12. The model presented in figure 8 has several aspects that should be improved:\na. It seems to imply that C. sakazakii is a gram positive (single membrane layer between\nextracellular /cytoplasm space)\nb. What is “inflammatory factor” in this model?\nc. Why have nitrate represented by both a green dot and also write out NO3-?\nd. The model should include the proposed mechanism whereby tungsten would inhibit\nthis pathway\ne. Explicitly incorporating host elements (e.g NOS via Nos2) into the model and its\ninteraction with C. sakazakii would be useful.\nResponse: Thank you for the comments. We have modified the Figure 8 according to\nyour suggestions. C. sakazakii is a Gram-negative bacteria, and we have showed the\ndouble membrane layer between extracellular /cytoplasm space. The “inflammatory\nfactor” in the original Fig. 8 has been changed into inducible nitric oxide synthase\n(iNOS) and its product, nitric oxide (NO). The representation of nitrate has been\nstandardized to its chemical formula (NO₃⁻). The tungsten inhibition pathway has been\nincluded in the revised Figure 8.\n13. Did the authors assess the impact of tungsten on their rat microbiomes? While\nprevious report suggested that tungsten may have minimal impact on mouse\nmicrobiotas, this should be validated in this rat model.\nResponse: Thank you for your suggestions. To investigate the effect of tungstate\ntreatment on the microbiota of neonatal rats, 2-day-old rats were treated with tungstate.\nAfter two days, DNA extracted from the caecal content of tungstate-treated rats and\ncontrol rats was analysed by high-throughput gene-sequencing of 16S rRNA. By\nanalyzing the alpha diversity, we found that Chao index and Shannon index between\nthe tungstate group and mock group exhibited no significant difference (Supplementary\nFig. 7F). The result of principal coordinate analysis (PCoA) using Bray-Curtis metric\ndistances of beta diversity also showed that tungstate treatment did not significantly\nalter the intestinal bacterial diversity (Supplementary Fig. 7G). Moreover, we assessed\nthe relative abundance of the gut microbiota in each group. Consistently, variations at\nthe genus level also showed that tungstate treatment did not alter the overall community\nstructure (Supplementary Fig. 7H-I). We have added these results and descriptions in\nlines 455-462 of the revised manuscript.\n14. On a similar line of thought, the authors tone down the statement that tungsten does\nnot adversely affect the commensal microbiota (line 458), as a single paper is being\nreferenced. This is likely an oversimplification as numerous bacteria employ\nmolybdate cofactors for and would be susceptible to the same tungsten-mediated\npoisoning. Moreover, any therapies for targeting C. sakazakii would naturally be employed in\ninfants who may be more susceptible to microbiota alterations given their young age.\nResponse: Thank you for the comments. As your concern above, we performed 16S\nrRNA sequencing on bacterial DNA from the caecal content of tungstate-treated and\ncontrol rats. The results showed that tungstate treatment did not significantly alter the\nintestinal bacterial diversity and community structure of the gut microbiota\n(Supplementary Fig. 7F-I). For details, please see the response to your Comment 13.\nIn addition, according to your comments, the sentence “Importantly, tungsten has been\nshown not to adversely affect commensal microbiota” has been changed to “ In light\nof both our work and prior evidence, tungsten appears to have a minimal effect on\ncommensal microbiota, though further validation is warranted” (line 524-526 of the\nrevised manuscript).\n15. Figure 5B shows an increase in nitrate production in RAW264.7 cell over time. Did\nthe authors do a mock infection to determine if nitrate is being induced by infection vs\naccumulating over time?\nResponse: Thank you for the comments. According to your suggestions, we\nremeasured nitrate levels in the supernatant of infected and un-infected macrophage\ncultures at 1, 4, 24 hpi to determine whether nitrate production is induced by infection\nor accumulates over time. The results showed a slight time-dependent increase in nitrate\nlevels in uninfected macrophages; however, the nitrate levels were significantly higher\nin macrophages infected with C. sakazakii at all time points. These data indicate that\ninfection of C. sakazakii induces the nitrate accumulation in macrophages. The relevant\nresult have been updated in Fig. 5B.\n16. The statement in lines 428-430 stating that “aerobic respiration” being dispensable\nfor virulence is to broad. Only single deletion mutants were tested and there could be\ncompensation between the pathways both in vivo and in vitro should the appropriate\nsubstrates be present.\nResponse: Thank you for your suggestions. We have now rewritten the sentence “our\nstudy found that aerobic respiration is dispensable for the virulence of C. sakazakii both\nin vivo and in vitro (Figs. 7A-C)” into “our study found that deletion of the cydAB,\nwhich encodes cytochrome bd oxidase, had no significant effect on the virulence of C.\nsakazakii both in vivo and in vitro (Fig. 7A-C)” in lines 494-496 of the revised\nmanuscript.\nReviewer #2 (Remarks to the Author):\nThe authors reported a signal transduction pathway centered on a novel sRNA, CsrN,\nwhich facilitates C. sakazakii in utilizing nitrate respiration in response to oxygen-\nlimited environments within the host, thereby enhancing its virulence in vivo. There\nwere some major concerns need to be addressed.\n1. Lines 21 and 54, C. sakazakii infections are usually associated with powdered infant\nformula, but the link with rice-based supplementary food remains contentious. The\nauthors should provide sufficient references—specifically clinical case reports with\nsupporting epidemiological data—to substantiate this assertion regarding rice-based\nproducts.\nResponse: Thank you for your valuable suggestions. According to the literature, C.\nsakazakii has been isolated in infant supplementary food in both China and Brazil over\nthe past five years (Food Res Int. 2020;137:109643. Microbiol Spectr.\n2022;10(5):e0119722). However, we did not find any reported clinical case attributed\nspecifically to rice-based supplementary food. Therefore, we revised the sentence to:\n“Although C. sakazakii is ubiquitous in the environment, its primary transmission\nsource is reconstituted powdered infant formula (PIF)” in lines 51-53 of the revised\nmanuscript.\n2. Line 145-150, It remains unclear whether the reported bacterial counts represent C.\nsakazakii exclusively or include other microorganisms. Please specify the\nselective/differential medium used for plating, as this critically impacts data\ninterpretation. Figure 2C-D presents survival rates and bioluminescent imaging for\nwild-type and ΔCsrN. To establish genetic causality, data for the complemented strain\n(ΔCsrN+pCsrN) must be included. This control is essential to demonstrate that\nobserved phenotypes result specifically from CsrN deletion rather than secondary\nmutations.\nResponse: Thank you for the comments. The bacteria strain used in this study carry\ndifferent antibiotic resistance, for instance, the WT strain spontaneously developed\nresistance to nalidixic acid; the mutant strains are resistant to chloramphenicol; and the\ncomplementary strains carry resistance corresponding to the plasmid they harbored. We\ndetermined the colony count of C. sakazakii by plating samples on antibiotic-containing\nagar.\nAs suggested, we have now performed additional experiments and included the\ncomplemented strains (ΔCsrN+pCsrN and ΔnarGHJI+pnarGHJI) in Fig. 2C-D and\n4C-D. The results demonstrated that complementation with CsrN or narGHJI restored\nthe systemic dissemination defects observed in the ΔCsrN and ΔnarGHJI, as\ndemonstrated by in vivo bioluminescent imaging (Fig. 2D and 4D). Consistently, the\npathogenicity of the complemented strains were restored to WT levels, as reflected by\nthe survival curves of infected rats (Fig. 2C and 4C). These results have been\nincorporated into Fig. 2C-D and 4C-D and lines 160-169, 268-272 of the revised\nmanuscript.\n3. Beyond the CGG motif in hairpin loop of CsrN, whether other nucleotides within the\ncomplementary region between 5′ UTR of narGHJI mRNA and the CsrN sRNA affect\ntheir binding interaction?\nResponse: Thank you for the comments. In bacteria, RNA stem-loops contributes to\nthe initial recognition events between RNAs, often facilitating hairpin loop-loop and\nloop-linear interactions (Nucleic Acids Res. 2010;38(10):3489-3501; Biochimie.\n2002;84(9):925-944). Within the stem-loop structure of RNA molecule, the stem is\ncomposed of base-paired nucleotide forming a stable double helix, whereas the loop is\nan unpaired region that forms a characteristic hairpin-like structure (Nat Commun. 2021\nMay 5;12(1):2530). Compared to the stem structure, the loop often provides a more\nflexible and accessible conformation for initiating molecular recognition, enabling\nspecific base-pairing interactions (Biochimie. 2002;84(9):925-944). In the case of CsrN,\nthe other nucleotides in CsrN which complemented with the 5′ UTR of narGHJI mRNA\nlargely formed the stem structure, making them less likely to be directly involved in\ntarget recognition. To investigate this further, we performed the Lux reporter assay\nusing ΔCsrN narGHJI-lux strain complementated with either wild-type CsrN or\nCsrNstem-mut (with CGGGACGGCAGCGC mutated to CGGGACAGCAGCGC, which\nhad a mutation in the stem region without disrupting the overall stem-loop structure).\nThe result showed that overexpression of CsrN or CsrNstem-mut in ΔCsrN narGHJI-lux\nstrain both significantly increased luminescence intensity (Response Fig. 4). It\nindicates that the complementary region between the stem region in CsrN and 5′ UTR\nof narGHJI mRNA is not essential for the direct recognition to the target. However, the\nstem region itself may provide a structure scaffold that facilitates the binding of the\nhairpin loop in CsrN to the 5′ UTR of narGHJI mRNA.\nResponse Fig. 4 Expression of the 5′ UTR of narGHJI-lux fusion when CsrN or CsrNstem-mut was\noverexpressed as indicated. n = 3 independent experiments.\n4. To establish that CsrN increases the expression of narGHJI by influencing its mRNA\nstability, the authors should perform genetic complementation experiments using a\nCsrN-expressing plasmid in ΔCsrN strains.\nResponse: Thank you for the comments. We have performed additional experiments\nand included the complemented strain (ΔCsrN+pCsrN) in Fig. 3N and O.\n5. Line 231-234, the survival of ΔCsrNΔnarGHJI exhibited no significant difference\nfrom that of ΔnarGHJI, the author speculated CsrN deletion did not affect the bacterial\nsurvival in macrophages in a ΔnarGHJI background. This experiment lacked the\nessential control group: the ΔCsrNΔnarGHJI strain complemented with CsrN\n(ΔCsrNΔnarGHJI + pCsrN).\nResponse: Thank you for your valuable suggestions. According to your suggestions,\nwe have now infected macrophages with WT, ΔCsrNΔnarGHJI or ΔCsrNΔnarGHJI\ncomplemented with CsrN (ΔCsrNΔnarGHJI + pCsrN) strain. We found that the\nsurvival of ΔCsrNΔnarGHJI and ΔCsrNΔnarGHJI + pCsrN in macrophages exhibited\nno significant difference, but both were significantly reduced compared to WT\n(Supplementary Fig. 4C), suggesting that CsrN deletion did not affect the bacterial\nsurvival in macrophages in a ΔnarGHJI background. Moreover, we also performed\ndisseminated infection assay in 2- to 5-day-old rat pups orally infected with WT,\nΔCsrNΔnarGHJI or ΔCsrNΔnarGHJI + pCsrN strains. The results showed that the\nsurvival rates and the bacterial burdens in the small intestine, colon, liver, spleen, and\nblood of rat pups infected with either ΔCsrNΔnarGHJI or ΔCsrNΔnarGHJI + pCsrN\nwere comparable, but both were significantly lower than those infected with WT\n(Supplementary Fig. 4E-F). These results confirmed that CsrN deletion did not affect\nthe systemic dissemination within the host in a ΔnarGHJI background. The relevant\nresults were updated in Supplementary Fig. 4C, 4E-F and lines 249-253, 273-280 of the\nrevised manuscript.\n6. The most significant limitation of this study is that, beyond comparing bacterial loads,\nit lacks supporting pathological evidence to substantiate the changes in C. sakazakii\nvirulence following gene knockout. Crucially, data demonstrating differences in key\nindicators of pathogenicity – such as cytopathy, cytotoxicity/cell damage, inflammatory\ncytokine levels, and histopathology of affected organs – are missing. Furthermore, the\nultimate cause of death in the infected mice remains undefined.\nWhile the ΔnarGHJI mutant group showed approximately 20% survival at 96 hours,\nall mice infected with the wild-type (WT) strain and the other knockout mutants (including\nthe ΔCsrNΔnarGHJI double mutant) succumbed to infection. The observed difference\nin time-to-death between WT-infected mice and knockout mutant-infected mice was\nonly about 24 hours. This relatively short time difference raises questions about the\nmagnitude and biological significance of the attenuation in virulence attributed to the\ngene knockouts.\nConsequently, while the study effectively demonstrates that CsrN and narGHJI\nknockouts impact the total bacterial load in vivo, it lacks direct evidence linking these\ngenetic modifications to a definitive change in C. sakazakii virulence.\nResponse: Thank you for your valuable suggestions. As recommended, we have now\ndetected the cytotoxicity in C. sakazakii infected macrophages, measured the level of\ninflammatory cytokines and performed histopathological analysis in intestinal tissues\nof infected rats. We found that the ΔCsrN and ΔnarGHJI strains were significantly less\ncytotoxic to macrophages compared to WT strain (Supplementary Fig. 2C and 4D).\nMoreover, the levels of TNF-α and IL-6 in the intestinal tissues of ΔCsrN- or\nΔnarGHJI-infected rats were significantly lower than those observed in WT-infected\nrats (Supplementary Fig. 2D and 4G). Histological examination revealed that intestinal\nsegments from WT-infected rats exhibited significantly higher histopathology scores\ncompared to those infected with ΔCsrN or ΔnarGHJI (Fig. 2E-F and 4E-F). These\nresults suggest that the regulation of narGHJI by CsrN promotes the survival of C.\nsakazakii in macrophages and enhances the systemic dissemination of bacteria in vivo,\nleading to increased disease intensity in the hosts. These results have been added in\nlines 146-151, 169-175, 253-257, 280-283 of the revised manuscript.\nReviewer #3 (Remarks to the Author):\nThis manuscript investigates a regulatory pathway in Cronobacter sakazakii that\nconnects the global regulator ArcA, the small RNA CsrN, and the nitrate reductase\noperon narGHJI. RNA-seq identifies narGHJI as a CsrN-upregulated operon. The\nauthors demonstrate that CsrN base pairs directly with the narG 5′UTR to stabilize its\nmRNA, and that ArcA binds to the csrN promoter to activate transcription. Evidence is\nprovided from different models linking this regulatory cascade to survival and\npathogenesis. Finally, they show that sodium tungstate, a nitrate reductase inhibitor,\nreduces bacterial burden and prolongs host survival. A noteworthy strength is that the\nphenotypes are clearly observed in a ΔcsrN strain. For many sRNAs, only\noverexpression reveals an effect, but here the loss-of-function phenotype is strong and\nreproducible. This underscores the biological importance of CsrN and strengthens the\nconclusions.\nOverall, the study is conceptually appealing: it defines a regulatory chain from a global\ntranscriptional regulator (ArcA), through an sRNA (CsrN), to a metabolic operon\n(narGHJI), establishing a direct link between regulatory RNA biology, metabolism, and\nvirulence. Nevertheless, several comments and questions, listed below, could further\nimprove the manuscript.\n1. Persistence and infection strategy\nThe data clearly support bacterial persistence in host niches—specifically survival in\nmacrophages and colonization of inflamed, oxygen-limited tissues—driven by nitrate\nrespiration. The manuscript uses “persistence” in this ecological/pathophysiological\nsense. However, antibiotic persistence (tolerance to bactericidal drugs) is not addressed,\neven though nitrosative stress is known to shape persistence in enteric pathogens.\nIncorporating antibiotic-challenge assays in infected cells would broaden the impact\nand connect the findings to the antibiotic persistence field.\nResponse: Thank you for your valuable suggestions. According to your suggestions,\nwe performed the C. sakazakii antibiotic survival assays in infected macrophages,\nfollowing a previously published protocol (Cell Host Microbe. 2023;31(6):993-\n1006.e6). Briefly, RAW264.7 cells were infected with WT, ΔCsrN and ΔnarGHJI\nstrains grown to the logarithmic phase at a multiplicity of infection (MOI) of 100. After\n45 minutes of incubation post-infection, a portion of the infected macrophages was used\nto assess bacterial invasion prior to antibiotic treatment. Intracellular bacteria were\nextracted by lysing the macrophages with 0.1 % Triton X-100 (Sigma), followed by\nten-fold serial dilutions in PBS and plating on LB agar to determine CFU counts at time\npoint T0. The remaining infected macrophages were treated with antibiotic by replacing\nthe medium with one supplemented with cefotaxime (100 μg/ml; TCI). At the indicated\ntime points, intracellular bacteria were again extracted as described above and plated\non LB agar to assess antibiotic survival. We found that the survival of ΔCsrN and\nΔnarGHJI showed no significant difference compared with the WT strain in\nmacrophages under antibiotic treatment (Response Fig. 5), suggesting that nitrate\nrespiration is dispensable for antibiotic persistence in C. sakazakii.\nResponse Fig. 5 Kinetics of cefotaxime survival of C. sakazakii WT, ΔCsrN or ΔnarGHJI strain\nin RAW264.7 cells. All values normalized to values after 45 min internalization (T0). n = 3\nindependent experiments.\n2. ΔarcA virulence phenotype\nThe ΔarcA strain is analyzed in depth for gene expression: ArcA binds the csrN\npromoter and is required for csrN and narGHJI induction under anaerobic conditions\nand during infection. While this firmly establishes transcriptional control, I did not see\ndirect in vivo virulence data for ΔarcA (e.g., reduced colonization or systemic spread\nin the rat model). Including ΔarcA infection data with complementation would fully\nclose the regulatory loop from sensor (ArcA) to virulence phenotype.\nResponse: Thank you for the comments. According to your comments, we have now\ninfected macrophages with WT, ΔarcA or ΔarcA complemented strain. We found that\nthe survival of ΔarcA in macrophages was significantly lower than WT and ΔarcA\ncomplemented strain (Supplementary Fig. 6M). Moreover, we also performed\ndisseminated infection assay in 2- to 5-day-old rat pups orally infected with WT, ΔarcA\nor ΔarcA complemented strain. The results showed that at 48 hpi, the bacterial burdens\nin the small intestine, colon, liver, spleen, and blood of rat pups infected with ΔarcA\nwere significantly lower than those in rats infected with WT or ΔarcA complemented\nstrain (Fig. 6J). These results suggest that ArcA, which activates the nitrate respiration\nunder anaerobic conditions, contributes to the virulence of C. sakazakii both in vitro\nand in vivo. The relevant results have been included in Fig. 6J and Supplementary Fig.\n6M and lines 405-408 of the revised manuscript.\n3. Role of Hfq\nSince most sRNAs in enterobacteria require Hfq for stability and function, the role of\nHfq in C. sakazakii deserves clarification. Does Hfq bind and stabilize CsrN as in other\nEnterobacteriaceae? If this has not been directly tested, a brief discussion of this point\nwould help readers interpret the generalizability of the mechanism.\nResponse: Thank you for your valuable suggestions. In our study, we found that\ndeletion of hfq did not affect the expression and the stability of CsrN (Supplementary\nFig. 1B and 1C), indicating that CsrN functions in an Hfq-independent manner. This\nfinding is consistent with recent studies reporting a growing number of Hfq-\nindependent sRNAs in bacteria (Nature. 2023;618(7964):358-364; Mol Cells.\n2019;42(5):426-439; J Bacteriol. 2006;188(2):576-586). To explore the distribution of\nCsrN, we performed a homology search based on its nucleotide sequence. The results\nshowed that CsrN is only present in Cronobacter spp. These results suggest that CsrN\nis a specific, Hfq-independent sRNA that contributes to the virulence of Cronobacter.\n4. Conservation of CsrN\nThe manuscript shows that CsrN is conserved across C. sakazakii species. What about\nconservation in other Gram-negative bacteria? If CsrN or a homolog exists more\nbroadly, that could expand the significance of the findings.\nResponse: Thank you for your valuable suggestions. As responsed above, we\nperformed a sequence homology search, which showed that CsrN is only present in\nCronobacter spp. We have added this result in lines 131-132 of the revised manuscript.\n5. Pathogen lifestyle and infection model\nIs C. sakazakii considered a true intracellular pathogen? Are macrophages its preferred\nniche in vivo, or is the intracellular stage primarily a transient step during dissemination?\nThe transition in the text to macrophage assays is quite abrupt. A fuller justification of\nthe macrophage model’s relevance would help contextualize the intracellular findings.\nResponse: Thank you for the comments. C. sakazakii has not traditionally been\nconsidered a true intracellular pathogen. However, it is worth noting that many bacteria\npreviously classified as extracellular, such as Escherichia coli, Group A Streptococcu,\nNeisseria meningitides, and Pseudomonas aeruginosa, have been shown to reside and\nsurvive within host cells under certain conditions (Mol Microbiol. 2020;113(3):541-\n545). Similarly, C. sakazakii has demonstrated a remarkable ability to survive inside\nhost macrophages (J Immunol. 2009 Nov 15;183(10):6588-99; Food Microbiol. 2021\nDec;100:103851). Importantly, intracellular survival within macrophages is an essential\nstep for C. sakazakii to establish systemic dissemination, as the pathogen can evade the\nhost immune defense, penetrate the tissue barrier, and translocate to the bloodstream,\nliver, and spleen through macrophages (PNAS 2022;119(11):e2118002119; Microb\nPathog. 2022;169:105643; Microb Pathog. 2021;158:105064).\nWe have added this information in lines 66-69 of the revised manuscript.\n6. Choice of animal model\nThe rationale for using neonatal rats instead of mice is not fully explained. Is this a\nvalidated or preferential model for C. sakazakii infection? A short justification would\nhelp. In addition, the term “systemic infection” may be too strong for a gavage model\nwhere blood samples were not collected to quantify bacteremia. It would be clearer to\ndescribe dissemination based on colonization of distal organs.\nResponse: Thank you for your important comments. The use of neonatal rats in our\nstudy is based on well-established physiological and immunological differences\nbetween rats and mice that make rats a more relevant model for neonatal systemic\ninfections, including those caused by E. coli K1, which is a pathogen with similar\ninvasive properties to C. sakazakii (Redox Biol. 2023;59:102588; Nat Commun.\n2018;9(1):2296). As detailed in the referenced literature (J Vis Exp. 2014 Oct\n29;(92):e52018), neonatal rats closely resemble human infants in their susceptibility to\nsystemic infection via the gastrointestinal route, displaying strong age-dependent\nvulnerability. In contrast, mice are less suitable due to their rapid overwhelming by\nsystemic infection and lack of comparable age dependency. Importantly, α-defensins,\nkey antimicrobial peptides that protect against systemic invasion, are highly expressed\nin Paneth cells and neutrophils in both humans and rats, but not in mice (J Vis Exp.\ntogether with the neonatal rat’s established use in previous studies of C. sakazakii\npathogenesis, colonization, and systemic dissemination (e.g., J Infect Dis. 2008 Aug\n15;198(4):586-93; mSystems. 2019 Aug 6;4(4):e00305-19.), makes it a validated and\npreferential model for studying the virulence of C. sakazakii.\nWe agree that precision in terminology is crucial. In our results, as shown in the original\nmanuscript, we collected blood samples and quantified bacteremia (Fig. 2B, 4B, and\n5G), which supports the use of \"systemic infection\" to describe the presence of\nbacteria in the circulatory system. However, we acknowledge that the primary focus of\nour analysis was on bacterial load in distal organs. Therefore, we have replaced the term\n\"systemic infection\" with \"systemic dissemination\" throughout the revised manuscript."}, {"round": 2, "reviewer_comments": {"reviewer_1": "Thank you for responding to my comments and incorporating them into the manuscript. I have no further concerns.", "reviewer_2": "The authors have adequately addressed all my concerns. I recommend the acceptance of the revised manuscript.", "reviewer_3": "I am satisfied with the responses and the revisions made to the manuscript. The additional experiments and clarifications have effectively addressed my comments and have further strengthened the study. In particular, the new infection and antibiotic-survival data, expanded discussion of the infection model, and inclusion of ArcA virulence phenotypes improve the overall impact of the work. I am happy with the manuscript in its revised form."}, "author_response": ""}]

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