Dihydrolipoamide Acetyltransferase AceF Influences the Type III Secretion System and Resistance to Oxidative Stresses through RsmY/Z in Pseudomonas aeruginosa

Carbon metabolism plays an important role in bacterial physiology and pathogenesis. The type III secretion system (T3SS) of Pseudomonas aeruginosa is a virulence factor that contributes to acute infections. It has been demonstrated that bacterial metabolism affects the T3SS. Meanwhile, expression of T3SS genes is negatively regulated by the small RNAs RsmY and RsmZ. In this study, we studied the relationship between the dihydrolipoamide acetyltransferase gene aceF and the T3SS. Our results reveal an upregulation of RsmY and RsmZ in the aceF mutant, which represses the expression of the T3SS genes. Meanwhile, the aceF mutant is more tolerant to hydrogen peroxide. We demonstrate that the expression levels of the catalase KatB and the alkyl hydroperoxide reductase AhpB are increased in the aceF mutant. The simultaneous deletion of rsmY and rsmZ in the aceF mutant restored the expression levels of katB and ahpB, as well as bacterial susceptibility to hydrogen peroxide. Thus, we identify a novel role of AceF in the virulence and oxidative response of P. aeruginosa.


Introduction
Pseudomonas aeruginosa is a Gram-negative bacterial pathogen that causes various chronic and acute infections in humans [1]. The bacterium harbors an arsenal of virulence determinants that play important roles in infections [1]. The type III secretion system (T3SS) is a syringe-like complex that directly translocates effector proteins into the host cell cytosol, which leads to the malfunction or death of the cell [2,3]. The expression of the T3SS genes is activated under Ca 2+ depletion (e.g., by EGTA) or contact with host cells [2,3]. Four major T3SS effectors have been identified in P. aeruginosa, namely, ExoY, ExoT, ExoU and ExoS [2]. ExoU has been shown to function as a phospholipase, which directly damages the host cell membrane and causes rapid necrotic death [4]. P. aeruginosa strains with the ability to inject ExoU are usually associated with poor clinical outcomes and increased mortality rates [5].
The T3SS machinery and effector genes are regulated by the master regulator ExsA [6]. The exsA gene is located in an exsC-exsE-exsB-exsA operon. The transcription of the exsA gene is driven by its own promoter (P exsA ) and the exsC promoter (P exsC ), which are positively regulated by Vfr and ExsA, respectively [7][8][9]. In addition to transcriptional regulators, small RNAs (sRNAs) are involved in the regulation of T3SS genes [10]. The sRNAs RsmY and RsmZ repress the expression of the T3SS genes by sequestering RsmA, a posttranscriptional regulator that positively regulates the T3SS genes and represses the type VI secretion systems (T6SSs) and biofilm formation [11][12][13]. The expression of rsmY and rsmZ is controlled by the GacS-GacA two-component system [14], whose function is repressed by RetS and activated by LadS [15]. The sensor kinase GacS phosphorylates the response regulator GacA, which binds to the promoters of rsmY and rsmZ and activates their transcription [14]. RetS is a hybrid two-component regulator with a sensor kinase domain and two response regulator domains [16]. It has been demonstrated that RetS inhibits the GacS activity through three mechanisms: (1) the siphoning of phosphates from the catalytic domain of GacS; (2) the inhibition of GacS autophosphorylation through direct interaction; and (3) the dephosphorylation of the receiver domain of GacS [16][17][18][19]. A recent study identified host-derived mucin glycans as ligands of RetS. The presence of mucin glycans represses the GacS-GacA two-component system through RetS [20]. Meanwhile, the GacS-GacA two-component system is positively regulated by two hybrid histidine kinases, PA1611 and LadS. PA1611 directly binds to RetS, which counteracts the repressive effect of RetS on GacS [21]. LadS activates the GacS-GacA two-component system by directly phosphorylating GacS [15].
Metabolism plays important roles in bacterial virulence [22]. It has been demonstrated that tryptophan and histidine utilization interfere with the expression of the T3SS genes in P. aeruginosa [23,24]. The catabolite repression control protein Crc is required for the expression of the T3SS genes in P. aeruginosa [25], indicating a relationship between carbon metabolism and the T3SS. The function of Crc is antagonized by a small RNA (sRNA), CrcZ [26]. In our previous screening for T3SS-related carbon metabolism genes, we found that triosephosphate isomerase (TpiA) affects T3SS gene expression through CrcZ [22]. The isocitrate lyase is required for the expression of the T3SS genes under oxygen-limited conditions [25]. Dacheux et al. found that transposon insertions in genes encoding the pyruvate dehydrogenase (PDH) subunits AceE and AceF (previously called AceA and AceB) resulted in the defective expression of the T3SS genes upon Ca 2+ depletion, which was restored by expression of exsA in trans [27]. A mutation in the E1 subunit (AceA/AceE) of pyruvate dehydrogenase resulted in the defective expression of exoS in response to Ca 2+ depletion in a minimal medium containing 0.1% tryptone and glutamate [28]. In this study, we demonstrate that the dihydrolipoamide acetyltransferase AceF inversely influences the T3SS and bacterial tolerance to oxidative stress through RsmY/Z.

Bacterial Strains and Plasmids
The bacterial strains, primers and plasmids used in this study are shown in Table S1. All strains were cultured in Luria-Bertani (LB) broth with agitation at 200 rpm at 37 • C. Antibiotics were used as follows: for Escherichia coli, ampicillin at 100 µg/mL, tetracycline at 10 µg/mL; for P. aeruginosa, tetracycline at 50 µg/mL, carbenicillin at 150 µg/mL. Construction of the aceF gene deletion mutant in the wild-type Pseudomonas aeruginosa reference strain PA14 was performed as described previously [29]. A 1005 bp fragment and a 1152 bp fragment that are upstream and downstream of the aceF open reading frame were amplified by PCR, respectively. The PCR products were ligated to the plasmid pEX18Tc. The resultant plasmid was transferred into an E. coli conjugation donor strain S17-1 and then transferred to wild-type PA14 by conjugation. The PA14 strains with the plasmid integrated into the chromosome (single-crossover mutant) were selected by tetracycline. The single-crossover mutants were grown in LB overnight and plated on plates with 5% sucrose. The aceF deletion mutants (double-crossover mutants) were screened by PCR with primers targeting the coding region of aceF (Table S1). Other chromosomal gene mutations were generated in the same way. For the overexpression of aceF and exsA, the coding regions with their native ribosome binding sequences were amplified by PCR using the PA14 chromosomal DNA as the template with the primers shown in Table S1. The PCR products were cloned into the pUCP20 plasmid in which the transcription of the genes is driven by the tac promoter on the plasmid.

Cytotoxicity Assay
Bacterial cytotoxicity was measured by examining the survival of the A549 cells following P. aeruginosa infection. A549 cells were cultured in RPMI 1640 medium containing 10% (v/v) heat-inactivated fetal bovine serum (hiFBS), penicillin (100 mg/mL) and streptomycin (100 mg/mL) at 37 • C with 5% CO 2 . Then, 1 × 10 5 cells were seeded into each well of a 24-well plate 20 h before bacterial infection. Overnight bacterial cultures were diluted into fresh LB and cultured at 37 • C to an OD 600 of 1.0. Then, bacterial cells were washed once with PBS and resuspended in PBS. A549 cells were infected with the bacteria at a multiplicity of infection (MOI) of 50. After 3 h of infection, the medium was removed, and each well was washed with PBS twice and stained with 0.25% crystal violet at room temperature for 15 min. An amount of 0.2 mL 95% ethanol was added to each well, followed by incubation for 30 min at room temperature with gentle shaking. Each sample was then subjected to measurement for OD 590 using a Varioskan Flash multimode microplate reader (Thermo Scientific, Vantaa, Finland).

Murine Acute Pneumonia Model
The animal experiments were in accordance with Chinese National and Nankai University guidelines for animal research. The protocol was approved by the animal care and use committee of Nankai University College of Life Sciences with the permit number NK-04-2012. The animal facility in Nankai University College of Life Sciences has been approved by Tianjin Municipal Science and Technology Bureau with the permission number SYXK 2019-0003. The animal care and use committee of Nankai University College of Life Sciences has been authorized by the local (Tianjin) government to review and approve animal experiment protocols. The animal facility and protocols are inspected by the local government regularly. The murine acute pneumonia model was performed as previously described [30,31]. The bacteria were cultured to OD600 of 1.0. Bacterial cultures were washed twice with PBS and resuspended to 2 × 10 8 CFU/mL. BALB/c mice (6 weeks, female) were anesthetized with 90 µL of 7.5% chloral hydrate by intraperitoneal injection and then intranasally inoculated with 4 × 10 6 CFU bacteria. The mice were sacrificed 12 h post infection (hpi) by asphyxiation with carbon dioxide. The euthanasia of mice was performed following the guidelines from the American Veterinary Medical Association (AVMA) for the euthanasia of animals: 2020 Edition (https://www.avma.org/KB/Policies/Documents/euthanasia.pdf, accessed on 10 January 2022). Less than four mice were placed in a cage. Carbon dioxide was introduced with a flow rate of 3-4 L/min until the mice were unconscious. The death of each mouse was confirmed by respiratory and cardiac arrest. The lungs were isolated and homogenized in PBS containing 1% peptone. The homogenate was serially diluted in LB and then plated on LB plates. The plates were incubated at 37 • C for 20 h before colony counting.

Histology
After infection with the indicated strains (4 × 10 6 CFU/mouse) for 12 h, the lungs of mice were removed and fixed with 4% paraformaldehyde (Servicebio, Wuhan, China) for 12 h, followed by dehydration in ethanol. The lungs were then embedded in paraffin and sectioned, followed by staining with hematoxylin and eosin. The samples were observed with microscopy as previously described [32].

Real-Time qPCR
Bacteria were cultured overnight and then diluted in fresh LB until they reached an OD 600 of 1.0. Total RNA was isolated using the ZOMANBIO RNA Rapid Extraction Kit (Zomanbio, Beijing, China). cDNA was synthesized with reverse transcriptase and random primers (TaKaRa, Dalian, China). SYBR II green Supermix (TaKaRa, Dalian, China) was used to perform real-time qPCR (RT-qPCR). To ensure the reliability of the results, we used the housekeeping genes rpsL and PA1805 as the internal controls for each RT-qPCR assay [33,34].

β-Galactosidase Assay
Overnight bacterial cultures were diluted in LB and grown to an OD 600 of 1.0 at 37 • C. A 500 µL volume of the bacterial cultures was resuspended in Z-buffer (8.5 g/L NaH 2 PO 4 , 4.8 g/L Na 2 HPO 4 , 0.75 g/L KCl, 0.12 g/L MgSO 4 , and 0.35% (v/v) β-mercaptoethanol, pH 7.0). The OD 600 of the bacterial suspensions were measured. Then, 10 µL of 0.1% SDS and 10 µL of chloroform were added to the bacterial suspension and vortexed for 15 s. Then, the mixture was incubated at 37 • C immediately after adding 100 µL of 4 mg/mL orthonitrophenyl-galactopyranoside (ONPG) (BBI Life Sciences, Shanghai, China). When the color of the mixture turned yellow, the reaction was stopped by addition of a 500 µL volume of 1 M Na 2 CO 3 . OD 420 of the reaction mixtures was measured. The β-galactosidase activities (Miller units) = (1000 × OD 420 )/T/500/OD 600 ; T represents reaction time [35].

H 2 O 2 Susceptibility Assay
Bacteria were grown at 37 • C to an OD 600 of 1.0. The bacterial cells were washed twice with PBS. After resuspension in PBS, the bacteria were incubated in the presence or absence of 300 mM H 2 O 2 for 1 h at 37 • C. The bacterial survival rate = (live bacterial number with H 2 O 2 treatment)/(live bacterial number without H 2 O 2 treatment).

Mutation in the aceF Gene Reduces the Expression of Type III Secretion System Genes
In our previous screening for metabolic genes that influence bacterial cytotoxicity, we found that an aceF::Tn mutant displayed defective cytotoxicity [22]. AceF, AceE and LpdG form the pyruvate dehydrogenase that connects the glycolysis pathway and the tricarboxylic acid cycle (Figure 1). To verify the role of AceF in bacterial cytotoxicity, we constructed an aceF in-frame deletion mutant. The deletion of the aceF gene reduced the cytotoxicity, which was restored by complementation with an aceF gene (Figure 2A). In P. aeruginosa, the T3SS plays an important role in bacterial cytotoxicity. In agreement with the reduced cytotoxicity, the mutation of aceF reduced the expression of the T3SS genes, including the regulatory genes exsA and exsC, the structural gene pcrV and the effector genes exoU, exoT and exoY ( Figure 2B, Figure S1). Complementation with the aceF gene or the overexpression of the exsA gene restored the expression of these genes ( Figure 2B). These results suggest that AceF affects bacterial cytotoxicity by regulating the expression of the T3SS genes.

AceF Affects the Expression of the T3SS Genes through RsmY and RsmZ
Given the role of AceF in carbon metabolism, we suspected that CrcZ might be involved in the regulation of the T3SS in ∆aceF. The CrcZ level in the ∆aceF mutant was 1, Microorganisms 2022, 10, 666 6 of 14 which is 9-fold lower than that in the wild-type PA14 ( Figure S2). Since CrcZ antagonists the function of Crc that positively regulates the T3SS, the downregulation of CrcZ in the ∆aceF mutant is unlikely to cause the defective T3SS. We next examined the expression levels of RsmY and RsmZ. qRT-PCR revealed upregulation of RsmY and RsmZ in the ∆aceF mutant ( Figure 3A). By using transcriptional fusions between a lacZ reporter gene and the promoters of rsmY and rsmZ (PrsmY-lacZ and PrsmZ-lacZ), we found that the promoter activities of rsmY and rsmZ were enhanced in the ∆aceF mutant ( Figure 3B). To examine whether the upregulation of RsmY/Z leads to the repression of the T3SS, we deleted rsmY and rsmZ in the ∆aceF mutant, which restored the expression of exsA and exoU and bacterial cytotoxicity ( Figure 4A,B). However, compared to the ∆aceF∆rsmY/Z triple mutant, the pcrV mRNA levels were higher in the ∆rsmY/Z mutant in the absence and presence of EGTA, and the mRNA level of exsC was higher in the ∆rsmY/Z mutant in the presence of EGTA ( Figure 4B). These results indicate that AceF might regulates the transcription or stabilities of pcrV and exsC through an RsmY/Z-independent mechanism. Collectively, these results demonstrate that AceF affects the T3SS and cytotoxicity mainly through RsmY/Z. ** p < 0.01 by ANOVA.

AceF Affects the Expression of the T3SS Genes through RsmY and RsmZ
Given the role of AceF in carbon metabolism, we suspected that CrcZ volved in the regulation of the T3SS in ΔaceF. The CrcZ level in the ΔaceF m which is 9-fold lower than that in the wild-type PA14 ( Figure S2). Since CrcZ the function of Crc that positively regulates the T3SS, the downregulation o ΔaceF mutant is unlikely to cause the defective T3SS. We next examined th levels of RsmY and RsmZ. qRT-PCR revealed upregulation of RsmY and ΔaceF mutant ( Figure 3A). By using transcriptional fusions between a lacZ r and the promoters of rsmY and rsmZ (PrsmY-lacZ and PrsmZ-lacZ), we fo promoter activities of rsmY and rsmZ were enhanced in the ΔaceF mutant (F examine whether the upregulation of RsmY/Z leads to the repression of the leted rsmY and rsmZ in the ΔaceF mutant, which restored the expression exoU and bacterial cytotoxicity ( Figure 4A,B). However, compared to the Δa triple mutant, the pcrV mRNA levels were higher in the ΔrsmY/Z mutant in and presence of EGTA, and the mRNA level of exsC was higher in the Δrsm in the presence of EGTA ( Figure 4B). These results indicate that AceF might transcription or stabilities of pcrV and exsC through an RsmY/Z-independent Collectively, these results demonstrate that AceF affects the T3SS and cytotox through RsmY/Z.  The bacteria were grown in LB with or without 5 mM EGTA to an OD 600 of 1. The RNA levels of rsmY and rsmZ were determined by real-time PCR. (B) PA14, the ∆aceF mutant and the complemented strain containing the P rsmY -lacZ (left) and P rsmZ -lacZ (right) transcriptional fusion were cultured in LB with or without 5 mM EGTA to an OD 600 of 1, followed by determination of the β-galactosidase activity. Data shown represent mean ± standard error of mean from three samples. * p < 0.05; ** p < 0.01 by ANOVA. Figure 3. Mutation of the aceF gene upregulates rsmY and rsmZ. (A) The bacteria wer with or without 5 mM EGTA to an OD600 of 1. The RNA levels of rsmY and rsmZ wer by real-time PCR. (B) PA14, the ΔaceF mutant and the complemented strain containing (left) and PrsmZ-lacZ (right) transcriptional fusion were cultured in LB with or withou to an OD600 of 1, followed by determination of the β-galactosidase activity. Data sho mean ± standard error of mean from three samples. * p < 0.05; ** p < 0.01 by ANOVA.

Role of AceF in Bacterial Virulence in an Acute Pneumonia Model
Since the P. aeruginosa T3SS plays an essential role in acute infections, w the virulence of the ΔaceF mutant in a murine acute pneumonia model by ino

Role of AceF in Bacterial Virulence in an Acute Pneumonia Model
Since the P. aeruginosa T3SS plays an essential role in acute infections, we examined the virulence of the ∆aceF mutant in a murine acute pneumonia model by inoculating 4 × 10 6 CFU of bacteria into each mouse. A pathology analysis of the infected lungs showed that the mutation of aceF alleviated the host inflammatory response, which is in agreement with the defective T3SS in the ∆aceF mutant ( Figure 5A). However, the bacterial loads of the lungs infected by the ∆aceF mutant were similar to those infected by wild-type PA14 ( Figure 5B). These results indicate that mutations in aceF might enhance a mechanism that promotes the bacterial survival independent of the T3SS. To test our hypothesis, we compared the bacterial loads of a ∆exsA mutant and a ∆exsA∆aceF double mutant. The deletion of exsA significantly attenuated the bacterial virulence. At the dose of 4 × 10 6 CFU per mouse, both the ∆exsA and ∆exsA∆aceF mutants were cleared at 12 hpi (data not shown). Thus, we increased the dose to 4 × 10 8 CFU per mouse. The bacterial loads in the ∆exsA∆aceF mutant infected mice were higher than those infected by the ∆exsA mutant ( Figure 5C). Given that the P. aeruginosa T3SS preferentially targets bactericidal neutrophils in the acute pneumonia model [36][37][38], these results suggest that the T3SS-defective ∆aceF mutant might be more tolerant to the killing mechanisms of neutrophils.
Microorganisms 2022, 10, x FOR PEER REVIEW 10 6 CFU of bacteria into each mouse. A pathology analysis of the infected lungs that the mutation of aceF alleviated the host inflammatory response, which is in agr with the defective T3SS in the ΔaceF mutant ( Figure 5A). However, the bacterial l the lungs infected by the ΔaceF mutant were similar to those infected by wild-typ ( Figure 5B). These results indicate that mutations in aceF might enhance a mechani promotes the bacterial survival independent of the T3SS. To test our hypothesis, w pared the bacterial loads of a ΔexsA mutant and a ΔexsAΔaceF double mutant. The d of exsA significantly attenuated the bacterial virulence. At the dose of 4 × 10 6 C mouse, both the ΔexsA and ΔexsAΔaceF mutants were cleared at 12 hpi (data not s Thus, we increased the dose to 4 × 10 8 CFU per mouse. The bacterial loads in t sAΔaceF mutant infected mice were higher than those infected by the ΔexsA muta ure 5C). Given that the P. aeruginosa T3SS preferentially targets bactericidal neutro the acute pneumonia model [36][37][38], these results suggest that the T3SS-defectiv mutant might be more tolerant to the killing mechanisms of neutrophils.

Mutation of the aceF gene Increases Bacterial Tolerance to Hydrogen Peroxide
The generation of reactive oxygen species (ROS) is a major bactericidal mec of neutrophils [39,40]. P. aeruginosa strains that are defective in the oxidative st sponse have been shown to be attenuated in virulence [41,42]. We thus examined b survival following H2O2 treatment. Compared to wild-type PA14, the ΔaceF mut played a higher survival rate ( Figure 6A).

Mutation of the aceF gene Increases Bacterial Tolerance to Hydrogen Peroxide
The generation of reactive oxygen species (ROS) is a major bactericidal mechanism of neutrophils [39,40]. P. aeruginosa strains that are defective in the oxidative stress response have been shown to be attenuated in virulence [41,42]. We thus examined bacterial survival following H 2 O 2 treatment. Compared to wild-type PA14, the ∆aceF mutant displayed a higher survival rate ( Figure 6A). We next examined whether RsmY/Z are involved in the regulation of ahpB and katB in the ΔaceF mutant. The deletion of rsmY/Z reduced the expression levels of ahpB and katB ( Figure 6B) as well as bacterial survival following H2O2 treatment ( Figure 6D). These results demonstrate that the upregulation of RsmY/Z increases the expression of ahpB and katB and subsequent H2O2 tolerance in the ΔaceF mutant.

Discussion
In this study, we found that the mutation of the dihydrolipoamide acetyltransferase gene aceF in P. aeruginosa resulted in defective T3SS and enhanced H2O2 tolerance, which To understand the mechanism of the increased tolerance to H 2 O 2 , we examined the expression of genes involved in bacterial oxidative stress response, including the regulator gene oxyR, catalase genes katA and katB and alkyl hydroperoxide reductase genes ahpB and ahpC. The mutation of the aceF gene resulted in the upregulation of ahpB and katB and reduced the mRNA levels of katA and ahpC by 1.4-and 1.7-fold ( Figure 6B). The deletion of ahpB and katB reduced the H 2 O 2 tolerance of the ∆aceF mutant, which reduced the differences in survival between wild-type PA14 and the ∆aceF mutant from 26.8-fold to 2and 4.2-fold, respectively ( Figure 6C). These results indicate that the upregulation of ahpB and katB contributes to the increased H 2 O 2 tolerance of the ∆aceF mutant.
We next examined whether RsmY/Z are involved in the regulation of ahpB and katB in the ∆aceF mutant. The deletion of rsmY/Z reduced the expression levels of ahpB and katB ( Figure 6B) as well as bacterial survival following H 2 O 2 treatment ( Figure 6D). These results demonstrate that the upregulation of RsmY/Z increases the expression of ahpB and katB and subsequent H 2 O 2 tolerance in the ∆aceF mutant.

Discussion
In this study, we found that the mutation of the dihydrolipoamide acetyltransferase gene aceF in P. aeruginosa resulted in defective T3SS and enhanced H 2 O 2 tolerance, which is due to the upregulation of RsmY/Z. By using PrsmY-lacZ and PrsmZ-lacZ transcriptional fusions, we found that the activities of the rsmY/Z promoters were elevated in the ∆aceF mutant. The transcription of rsmY/Z is under the direct regulation of the two-component regulatory system GacS-GacA. However, we did not observe an upregulation of gacS or gacA in the ∆aceF mutant (data not shown). This warrants further studies to examine whether the activity of GacS-GacA is enhanced (e.g., phosphorylation of GacA) or whether other regulatory genes are involved in the regulation of rsmY/Z. P. aeruginosa produces catalases and alkyl hydroperoxide reductases to detoxify ROS [43]. The expression of these oxidative response genes is activated by the transcriptional regulator OxyR upon oxidative stresses [44]. OxyR is an LysR-type transcriptional regulator that consists of an N-terminal DNA-binding domain and a C-terminal regulatory domain. The presence of H 2 O 2 leads to the formation of an intramolecular disulfide bond in the C-terminal regulatory domain, which results in the activation of OxyR [45,46]. In the ∆aceF mutant, ahpB and katB were upregulated, and ahpC and katA were slightly downregulated. However, the mRNA levels of oxyR were similar to wild-type PA14. These results suggest that AceF is unlikely to influence the protein level or function of OxyR. Since the deletion of rsmY and rsmZ reduced the expression levels of ahpB and katB, it might be possible that an unknown regulatory gene is involved in the regulation of rsmY and rsmZ and subsequent ahpB and katB in the ∆aceF mutant. In addition to OxyR, the stress sigma factor RpoS positively regulates the catalase genes katA and katB [47,48]. In Pseudomonas fluorescens, the expression of rpoS is activated by GacA and repressed by RsmA [49]. Further studies are warranted to examine whether the mutation of aceF affects the level of RpoS. The stringent response has been shown to control the expression of the catalase genes in P. aeruginosa and promotes the bacterial survival under oxidative stresses [47,50]. The stringent response is controlled by the molecule guanosine tetra-and penta-phosphate (p)ppGpp [51]. The homeostasis of (p)ppGpp is controlled by two enzymes, namely RelA and SpoT. RelA is a (p)ppGpp synthetase that is activated by amino acid starvation. SpoT functions as both (p)ppGpp synthetase and hydrolase [52]. It has been demonstrated that in E. coli, acyl carrier protein (Acyl-ACP) activates the (p)ppGpp hydrolysis activity of SpoT, whereas uncharged ACP activates the (p)ppGpp synthetase activity [53], indicating a connection between carbon metabolism and the stringent response. The pyruvate dehydrogenase connects the glycolysis and the TCA cycle by converting pyruvates to acetyl-CoA that influx into the TCA cycle. Thus, the mutation of aceF might impede the TCA cycle, which might affect the activity of SpoT. Further studies are needed to examine whether the stringent response is activated in the ∆aceF mutant.
Previously, we found that the mutation of another carbon metabolism gene eno results in the downregulation of ahpB and ahpC, which increases bacterial susceptibility to oxidative stresses [32]. The eno encoded enolase is a glycolytic enzyme that reversibly catalyzes the dehydration of 2-phosphoglycerate to phosphoenolpyruvate [54]. In addition, enolase interacts with ribonuclease (RNaseE), polynucleotide phosphorylase (PNPase) and the RNA helicase RhlB to form the RNA degradosome, which plays important roles in RNA processing and degradation [55,56]. Further studies are warranted to elucidate whether the downregulation of ahpB and ahpC is due to defective assembly of the RNA degradosome or carbon metabolism.
Overall, our results demonstrated that the mutation of aceF induces the expression of the sRNA RsmYZ, which represses the expression of T3SS genes and increases bacterial tolerance to H 2 O 2 . Previous and current studies have revealed that interruptions of carbon metabolism in different reactions result in distinct expression patterns of bacterial virulence factors, demonstrating a complex relationship between bacterial metabolism and virulence.

Conflicts of Interest:
The authors declare no conflict of interest.