Risk factors and clinical features for pulmonary paragonimiasis-associated pneumothorax.

BACKGROUND: Pulmonary paragonimiasis, a food-borne zoonotic helminthiasis, is a parasitic disease of the lung caused by infection with trematodes species of the genus Paragonimus. Although pneumothorax has been reported as occuring with paragonimiasis, to date no study has been performed concerning the clinical features and predictive risk factors for this condition. METHODS: This retrospective study, which aims to fill this gap, was conducted at Jeonbuk National University Hospital. All patients (aged ≥19 years) were diagnosed with paragonimiasis between May 2011 and December 2021. Medical records were reviewed and information concerning age, sex, vital signs, underlying diseases, clinical signs and symptoms, laboratory findings, radiologic findings, treatment, and clinical outcomes was collected. An odds ratio (OR) for the risk factors associated with pneumothorax was calculated using the binary logistic regression model. RESULTS: Among 179 consecutive patients diagnosed with pulmonary paragonimiasis, the postive rate of pneumothorax was 10.6% (19/179). Pneumothorax occurred mostly in the right lung (78.9%, 15/19), and intrapulmonary parenchymal lesions showed an ipsilateral relationship with pneumothorax (94.7%, 18/19). Fifteen patients (78.9%, 15/19) of pneumothorax associated with pulmonary paragonimiasis are accompanied by pleural effusion. Most of patients with pneumothorax (89.5%, 17/19) underwent chest tube insertion as a first treatment. Three patients (15.8%) showed relapses but in no case was a death recorded. Asthma (odds ratio [OR] 8.10, 95% confidence interval [CI] 1.43-45.91), chest pain (OR 8.15, 95% CI 2.70-24.58), and intrapulmonary lesions (OR 8.94, 95% CI 1.12-71.36) were independent risk factors for pulmonary paragonimiasis-associated pneumothorax. CONCLUSIONS: Our findings suggest that clinicians should keep in mind the possibility of pneumothorax when approached by patients with pulmonary paragonimiasis complaining of chest pain, accompanied by intrapulmonary lesions or with asthma as an underlying disease.

AREL1 resists the apoptosis induced by TGF-ß by inhibiting SMAC in vascular endothelial cells.

Abnormal apoptosis of vascular endothelial cells is an important feature of arteriosclerosis (AS). Here, we induced apoptosis in human umbilical vein endothelial cells (HUVECs) using transforming growth factor-ß (TGF-ß), and investigated the role of antiapoptotic E3 ubiquitin ligase (AREL1) in the apoptosis of vascular endothelial cells. We proved that AREL1 is downregulated in TGF-ß treated HUVECs. The overexpression of AREL1 inhibits the activation of Caspase-3 and Caspase-9 and attenuates cell apoptosis induced by TGF-ß. According to the result of coimmunoprecipitation, AREL1 interacts with the proapoptotic proteins the second mitochondria-derived activator of caspases (SMAC) in TGF-ß treated HUVECs. In addition, miR-320b inhibits the expression of AREL1, and the overexpression of AREL1 attenuates the apoptosis induced by miR-320b mimics in HUVECs. In conclusion, AREL1 is downregulated by miR-320b. AREL1 overexpression inhibits TGF-ß induced apoptosis through downregulating SMAC in vascular endothelial cells. Our study explores pathogenesis regulation mechanism and new biological therapeutic targets for vascular disease.

Carrier-Free Immobilization of Multi-Enzyme Complex Facilitates In†Vitro Synthetic Enzymatic Biosystem for Biomanufacturing.

A method is developed for carrier-free immobilization of multi-enzyme complexes with more than four enzymes by utilization of polypeptide interactions (SpyCatcher-SpyTag and dockerin-cohesin) and enzyme component self-oligomerization. Two pairs of scaffoldins with different arrangements of SpyCatcher-SpyTag and cohesins are prepared to recruit the four dockerin-containing cascade enzymes (i. e., alpha-glucan phosphorylase, phosphoglucomutase, inositol 1-phosphate synthase, and inositol 1-phosphatase) that can convert starch into inositol, forming multi-enzyme complexes. These self-assembled enzyme complexes show higher initial reaction rates than the four-enzyme cocktail. Moreover, water-insoluble self-assembled multi-enzyme complexes are observed, being the carrier-free immobilized multi-enzyme complex aggregates. These immobilized enzyme complexes can be recycled easily by simple centrifuging followed by resuspension for another round of reaction. Not only can these immobilized enzyme complexes be obtained by mixing the purified enzyme components, but also by the mixing of crude cell extracts. Therefore, the strategy for the carrier-free immobilization of enzyme complex sheds light on improving the catalytic capability of in vitro synthetic enzymatic biosystems.

A thermophilic phosphatase from Methanothermobacter marburgensis and its application to in vitro biosynthesis.

Phosphatases catalyze the irreversible dephosphorylation of phosphate-containing compounds, and hence can be applied as the final enzymatic step for the synthesis of various biochemicals. However, the extensive substrate spectrums of phosphatases impose a great challenge for efficient biomanufacturing. Characterization of phosphatases is therefore of extreme importance. In this study, MmPase, a putative HAD phosphatase from Methanothermobacter marburgensis, was expressed, purified, and characterized. Recombinant MmPase was readily expressed in Escherichia coli, and required metal ions such as Mn2+ or Mg2+ to function. MmPase worked optimally at 50 °C, pH 6.5, and exhibited a half-life of 6.5 h under this condition. Among all substrates tested, MmPase established the highest dephosphorylation activity against D-tagatose 6-phosphate, and was relatively specific for this substrate than for D-glucose 1-phosphate, D-glucose 6-phosphate, and D-fructose 6-phosphate. Therefore, MmPase was integrated into an in vitro synthetic enzymatic biosystem for the one-pot production of D-tagatose from maltodextrin, and achieved a product yield of 37.6%. Our studies of MmPase provided a promising strategy for the economic and efficient production of D-tagatose in the future.

[Advances in a new energy system based on electricity-hydrogen-carbohydrate cycle].

The development of green and low-carbon renewable energy systems has become an important international consensus. It is also an essential path for China to implement the dual-carbon strategy, ensure national energy security, and achieve sustainable development. This review introduces the theory of a new energy system based on electricity-hydrogen-carbohydrate (EHC) cycle, and highlights the biotransformations of carbohydrate/water-to-hydrogen, carbohydrate-to-electricity, and CO2-to-carbohydrate powered by hydrogen- or electric-energy based on the in vitro synthetic enzymatic biosystems (ivSEB) developed by Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences in the past decade. We elaborate the design principle and the molecular basis of ivSEB, and further expand from the EHC cycle to in vitro biomanufacturing with starch as the feedstock. Combined with the latest research advances, we analyze and discuss advantages and disadvantages of ivSEB, prospect future directions, so as to promote the green, low-carbon and sustainable development of economy and society.

Curcumin alleviates acute kidney injury in a dry-heat environment by reducing oxidative stress and inflammation in a rat model.

Curcumin exhibits anti-inflammatory and antioxidant activities. We investigated the protective effects of curcumin in a renal injury rat model under dry-heat conditions. We divided Sprague-Dawley rats into four groups: dry-heat 0- (normal temperature control group), 50-, 100-, and 150-minute groups. Each group was divided into five subgroups (n = 10): normal saline (NS), sodium carboxymethylcellulose (CMCNa), and curcumin pretreated low, medium, and high-dose (50, 100, and 200 mg/kg, respectively) groups. Compared to the normal temperature group, serum creatinine, blood urea nitrogen, urinary kidney injury molecule-1, and neutrophil gelatinase-associated load changes in lipoprotein (NGAL) levels were significantly increased in the dry-heat environment group (P < .05); inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) expression and malondialdehyde (MDA) and related inflammatory factor levels were increased in the kidney tissue. Superoxide dismutase (SOD) and catalase (CAT) levels were decreased. However, following all curcumin pretreatment, the serum levels of kidney injury indicators and NGAL were decreased in the urine compared to those in the NS and CMCNa groups (P < .05), whereas renal SOD and CAT activities were increased and MDA was decreased (P < .05). Renal tissues of the 150-minute group showed obvious pathological changes. Compared to the NS group, pathological changes in the renal tissues of the 100- and 200-mg/kg curcumin groups were significantly reduced. Furthermore, iNOS and COX-2 expression and inflammatory factor levels were decreased after curcumin treatment. Curcumin exerted renoprotective effects that were likely mediated by its antioxidant and anti-inflammatory effects in a dry-heat environment rat model.

High-efficiency transformation of archaea by direct PCR products with its application to directed evolution of a thermostable enzyme.

Hyperthermophilic archaea with unique biochemical and physiological characteristics are important organisms for fundamental research of life science and have great potential for biotechnological applications. However, low transformation efficiency of foreign DNA molecules impedes developments in genetic modification tools and industrial applications. In this study, we applied prolonged overlap extension PCR (POE-PCR) to generate multimeric DNA molecules and then transformed them into two hyperthermophilic archaea, Thermococcus kodakarensis KOD1 and Pyrococcus yayanosii A1. This study was the first example to demonstrate the enhanced transformation efficiencies of POE-PCR products by a factor of approximately 100 for T. kodakarensis KOD1 and 8 for P. yayanosii A1, respectively, relative to circular shuttle plasmids. Furthermore, directed evolution of a modestly thermophilic enzyme, Methanothermococcus okinawensis 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR), was conducted to obtain more stable ones due to high transformation efficiency of T. kodakarensis (i.e. ~3 × 104  CFU per µg DNA). T. kodakarensis harbouring the most thermostable MoHMGR mutant can grow in the presence of a thermostable antibiotic simvastatin at 85°C and even higher temperatures. This high transformation efficiency technique could not only help develop more hyperthermophilic enzyme mutants via directed evolution but also simplify genetical modification of archaea, which could be novel hosts for industrial biotechnology.

The stringent response factor, RelA, positively regulates T6SS4 expression through the RovM/RovA pathway in Yersinia pseudotuberculosis.

The type VI secretion system (T6SS) is a versatile molecular machinery widely distributed in Gram-negative bacteria. The activity of the T6SS is tightly regulated by various mechanisms, including quorum sensing (QS), iron concentration, and transcriptional regulators. Here we demonstrated that the stringent response regulator, RelA, contributes to bacterial resistance to multiple environmental stresses in Yersinia pseudotuberculosis. We also revealed that the stress resistance function of stringent response (SR) was partially mediated by the general stress response T6SS4 system. RelA positively regulates the expression of T6SS4 to combat various stresses in response to nutrition starvation collectively mediated by the RovM and RovA regulators. These findings revealed not only the important role of T6SS4 in SR induced stress resistance, but also a new pathway to regulate T6SS4 expression in response to starvation stress.

A Recombinant 12-His Tagged Pyrococcus furiosus Soluble [NiFe]-Hydrogenase I Overexpressed in Thermococcus kodakarensis KOD1 Facilitates Hydrogen-Powered in vitro NADH Regeneration.

Soluble hydrogenase I (SHI) from the hyperthermophilic archaeon Pyrococcus furiosus is a heterotetrameric [NiFe] hydrogenase that catalyzes the reversible reduction of protons by NADPH into hydrogen gas (H2 ). Here, the authors expressed the four αßγδ subunits of SHI encoded by one gene cluster in another hyperthermophilic archaeon, Thermococcus kodakarensis KOD1, which uses its hydrogenase maturation apparatus without the coexpression of native P. furiosus hydrogenase endopeptidases (maturation proteases). The SHI overexpression of T. kodakarensis resulted in more than 1200-fold enhancement in the hydrogenase activity of the cell lysate compared to that of the host strain with an empty vector. An active, purified 12-His tagged recombinant SHI (rSHI) is obtained by one-step affinity adsorption on nickel-charged resin. Size-exclusion chromatography show that purified rSHI is heterotetrameric and has a molecular mass of 150 kDa. The purified rSHI has a half-life of 70 h at 80 °C. This rSHI is used to design a novel in vitro synthetic enzymatic biosystem to convert pyruvate and H2 gas into lactate in a theoretical yield, whereas rSHI is used for NADPH regeneration; an FMN-containing diaphorase (DI) is used to match NADP-preferred SHI and NAD-preferred lactate dehydrogenase (LDH). This study provides a cost-efficient method to obtain hyperthermostable hydrogenases, which can be used in in vitro synthetic enzymatic biosystems for cofactor regeneration and hydrogen production.

RovM and CsrA Negatively Regulate Urease Expression in Yersinia pseudotuberculosis.

Urease acts as an important acid resistance system and virulence factor that is widespread among microorganisms. RovM is a global regulator that regulates a series of genes and pathways including acid survival systems in the enteric bacterium Yersinia pseudotuberculosis (Yptb). However, whether RovM regulates the urease activity in Yptb was still unknown. In this study, by using qualitative and quantitative urease assays, we show that the urease expression responds to nutrient conditions and the RovM protein represses urease expression by binding to its promoter. A previously reported positive regulator OmpR activates urease activity but RovM plays a dominant role in different nutrient conditions. In addition, carbon storage regulator system A (CsrA), the upstream regulator of RovM, dramatically down-regulates urease activity possibly by its binding to the Shine-Dalgarno (SD) sequence of the mRNA encoding the urease. In conclusion, this study demonstrates that urease activity is strictly controlled by nutrient conditions and is down-regulated by the CsrA-RovM pathway.

A starvation-induced regulator, RovM, acts as a switch for planktonic/biofilm state transition in Yersinia pseudotuberculosis.

The transition between the planktonic state and the biofilm-associated state is a key developmental decision for pathogenic bacteria. Biofilm formation by Yersinia pestis is regulated by hmsHFRS genes (ß-1, 6-N-acetyl-D-glucosamine synthesis operon) in its flea vector and in vitro. However, the mechanism of biofilm formation in Yersinia pseudotuberculosis remains elusive. In this study, we demonstrate that the LysR-type regulator RovM inversely regulates biofilm formation and motility in Y. pseudotuberculosis by acting as a transcriptional regulator of these two functions. RovM is strongly induced during growth in minimal media but strongly repressed in complex media. On one hand, RovM enhances bacterial motility by activating the expression of FlhDC, the master regulator of flagellar genes, via the recognition of an operator upstream of the flhDC promoter. On the other hand, RovM represses ß-GlcNAc production under nutrition-limited conditions, negatively regulating hmsHFRS expression by directly binding to the -35 element of its promoter. Compared to wild-type bacteria, the rovM mutant established denser biofilms and caused more extensive mortality in mice and silkworm larvae. These results indicate that RovM acts as a molecular switch to coordinate the expression of genes involved in biofilm formation and motility in response to the availability of nutrients.

The dual transcriptional regulator RovM regulates the expression of AR3- and T6SS4-dependent acid survival systems in response to nutritional status in Yersinia pseudotuberculosis.

Coordinated regulation of various acid survival systems in response to environmental stimuli is crucial for the adaptation of enteropathogenic bacteria to acidic environments such as the stomach. In this study, we demonstrated that the RovM protein, a central regulator of the CsrABC-RovM-RovA cascade, conversely regulates the expression of two acid survival systems in Yersinia pseudotuberculosis by acting as a dual transcriptional regulator. RovM activated the expression of T6SS4, which is essential for bacterial survival under mild acidic conditions, by binding upstream of the T6SS4 promoter. On the contrary, RovM repressed the expression of a functional arginine-dependent acid resistance system (AR3), which is crucial for bacterial survival under strong acidic conditions, by directly binding to the -35 element in the AR3 promoter. Consistent with previous findings that rovM expression responds to the availability of nutrients, the expression of T6SS4 and AR3 was differentially regulated by nutritional status. Based on these results, a dynamic model whereby RovM coordinately regulates the expression of AR3 and T6SS4 in response to the availability of nutrients in the environment was proposed.

Roles of RpoS in Yersinia pseudotuberculosis stress survival, motility, biofilm formation and type VI secretion system expression.

RpoS (σ(S)), the stationary phase/stress σ factor, controls the expression of a large number of genes involved in cellular responses to a variety of stresses. However, the role of RpoS appears to differ in different bacteria. While RpoS is an important regulator of flagellum biosynthesis, it is associated with biofilm development in Edwardsiella tarda. Biofilms are dense communities formed by bacteria and are important for microbe survival under unfavorable conditions. The type VI secretion system (T6SS) discovered recently is reportedly associated with several phenotypes, ranging from biofilm formation to stress sensing. For example, Vibrio anguillarum T6SS was proposed to serve as a sensor for extracytoplasmic signals and modulates RpoS expression and stress response. In this study, we investigated the physiological roles of RpoS in Yersinia pseudotuberculosis, including bacterial survival under stress conditions, flagella formation, biofilm development and T6SS expression. We found that RpoS is important in resistance to multiple stressors-including H2O2, acid, osmotic and heat shock-in Y. pseudotuberculosis. In addition, our study showed that RpoS not only modulates the expression of T6SS but also regulates flagellum formation by positively controlling the flagellar master regulatory gene flhDC, and affects the formation of biofilm on Caenorhabditis elegans by regulating the synthesis of exopolysaccharides. Taken together, these results show that RpoS plays a central role in cell fitness under several adverse conditions in Y. pseudotuberculosis.

Type VI Secretion System Transports Zn2+ to Combat Multiple Stresses and Host Immunity.

Type VI secretion systems (T6SSs) are widespread multi-component machineries that translocate effectors into either eukaryotic or prokaryotic cells, for virulence or for interbacterial competition. Herein, we report that the T6SS-4 from Yersinia pseudotuberculosis displays an unexpected function in the transportation of Zn2+ to combat diverse stresses and host immunity. Environmental insults such as oxidative stress induce the expression of T6SS-4 via OxyR, the transcriptional factor that also regulates many oxidative response genes. Zinc transportation is achieved by T6SS-4-mediated translocation of a novel Zn2+-binding protein substrate YezP (YPK_3549), which has the capacity to rescue the sensitivity to oxidative stress exhibited by T6SS-4 mutants when added to extracellular milieu. Disruption of the classic zinc transporter ZnuABC together with T6SS-4 or yezP results in mutants that almost completely lost virulence against mice, further highlighting the importance of T6SS-4 in resistance to host immunity. These results assigned an unconventional role to T6SSs, which will lay the foundation for studying novel mechanisms of metal ion uptake by bacteria and the role of this process in their resistance to host immunity and survival in harmful environments.

Engineering an enhanced, thermostable, monomeric bacterial luciferase gene as a reporter in plant protoplasts.

The application of the luxCDABE operon of the bioluminescent bacterium Photorhabdus luminescens as a reporter has been published for bacteria, yeast and mammalian cells. We report here the optimization of fused luxAB (the bacterial luciferase heterodimeric enzyme) expression, quantum yield and its application as a reporter gene in plant protoplasts. The fused luxAB gene was mutated by error prone PCR or chemical mutagenesis and screened for enhanced luciferase activity utilizing decanal as substrate. Positive luxAB mutants with superior quantum yield were subsequently shuffled by DNase I digestion and PCR assembly for generation of recombinants with additional increases in luciferase activity in bacteria. The coding sequence of the best recombinant, called eluxAB, was then optimized further to conform to Arabidopsis (Arabidopsis thaliana) codon usage. A plant expression vector of the final, optimized eluxAB gene (opt-eluxAB) was constructed and transformed into protoplasts of Arabidopsis and maize (Zea mays). Luciferase activity was dramatically increased for opt-eluxAB compared to the original luxAB in Arabidopsis and maize cells. The opt-eluxAB driven by two copies of the 35S promoter expresses significantly higher than that driven by a single copy. These results indicate that the eluxAB gene can be used as a reporter in plant protoplasts. To our knowledge, this is the first report to engineer the bacterium Photorhabdus luminescens luciferase luxAB as a reporter by directed evolution which paved the way for further improving the luxAB reporter in the future.

Gene expression analysis of potential genes and pathways involved in the pathogenic mechanisms of parvovirus B19 in human colorectal cancer.

In order to investigate the pathogenic mechanisms of parvovirus B19 in human colorectal cancer, plasmids containing the VP1 or VP2 viral capsid proteins or the NS1 non-structural proteins of parvovirus B19 were constructed and transfected into primary human colorectal epithelial cells and LoVo cells. Differential gene expression was detected using a human genome expression array. Functional gene annotation analyses were performed using Database for Annotation, Visualization and Integrated Discovery v6.7 software. Gene ontology (GO) analyses revealed that VP1-related functions included the immune response, immune system process, defense response and the response to stimulus, while NS1-associated functions were found to include organelle fission, nuclear division, mitosis, the M-phase of the mitotic cell cycle, the mitotic cell cycle, M-phase, cell cycle phase, cell cycle process and cell division. Pathway expression analysis revealed that VP1-associated pathways included cell adhesion molecules, antigen processing and presentation, cytokines and the inflammatory response. Moreover, NS1-associated pathways included the cell cycle, pathways in cancer, colorectal cancer, the wnt signaling pathway and focal adhesion. Among the differential genes detected in the present study, 12 genes were found to participate in general cancer pathways and six genes were observed to participate in colorectal cancer pathways. NS1 is a key molecule in the pathogenic mechanism of parvovirus B19 in colorectal cancer. Several GO categories, pathways and genes were selected and may be the key targets through which parvovirus B19 participates in colorectal cancer pathogenesis.

Bioluminescence resonance energy transfer system for measuring dynamic protein-protein interactions in bacteria.

UNLABELLED: Protein-protein interactions are important for virtually every biological process, and a number of elegant approaches have been designed to detect and evaluate such interactions. However, few of these methods allow the detection of dynamic and real-time protein-protein interactions in bacteria. Here we describe a bioluminescence resonance energy transfer (BRET) system based on the bacterial luciferase LuxAB. We found that enhanced yellow fluorescent protein (eYFP) accepts the emission from LuxAB and emits yellow fluorescence. Importantly, BRET occurred when LuxAB and eYFP were fused, respectively, to the interacting protein pair FlgM and FliA. Furthermore, we observed sirolimus (i.e., rapamycin)-inducible interactions between FRB and FKBP12 and a dose-dependent abolishment of such interactions by FK506, the ligand of FKBP12. Using this system, we showed that osmotic stress or low pH efficiently induced multimerization of the regulatory protein OmpR and that the multimerization induced by low pH can be reversed by a neutralizing agent, further indicating the usefulness of this system in the measurement of dynamic interactions. This method can be adapted to analyze dynamic protein-protein interactions and the importance of such interactions in bacterial processes such as development and pathogenicity. IMPORTANCE: Real-time measurement of protein-protein interactions in prokaryotes is highly desirable for determining the roles of protein complex in the development or virulence of bacteria, but methods that allow such measurement are not available. Here we describe the development of a bioluminescence resonance energy transfer (BRET) technology that meets this need. The use of endogenous excitation light in this strategy circumvents the requirement for the sophisticated instrument demanded by standard fluorescence resonance energy transfer (FRET). Furthermore, because the LuxAB substrate decanal is membrane permeable, the assay can be performed without lysing the bacterial cells, thus allowing the detection of protein-protein interactions in live bacterial cells. This BRET system added another useful tool to address important questions in microbiological studies.

FliS modulates FlgM activity by acting as a non-canonical chaperone to control late flagellar gene expression, motility and biofilm formation in Yersinia pseudotuberculosis.

The FlgM-FliA regulatory circuit plays a central role in coordinating bacterial flagellar assembly. In this study, we identified multiple novel binding partners of FlgM using bacterial two-hybrid screening. Among these binding partners, FliS, the secretion chaperone of the filament protein FliC, was identified to compete with FliA for the binding of FlgM. We further showed that by binding to FlgM, FliS protects it from secretion and degradation, thus maintaining an intracellular pool of FlgM reserved as the FliS-FlgM complex. Consequently, we found that the flagellar late-class promoter activities are significantly increased in the fliS deletion mutant. The fliS mutant is weakly motile and shows significantly increased biofilm formation on biotic surface. Based on the results obtained, we established for the first time the regulatory role of the flagellin chaperone FliS to fine-tune late flagellar assembly by modulating FlgM activity.

A type VI secretion system regulated by OmpR in Yersinia pseudotuberculosis functions to maintain intracellular pH homeostasis.

Type VI secretion systems (T6SSs) which widely distributed in Gram-negative bacteria have been primarily studied in the context of cell interactions with eukaryotic hosts or other bacteria. We have recently identified a thermoregulated T6SS4 in the enteric pathogen Yersinia pseudotuberculosis. Here we report that OmpR directly binds to the promoter of T6SS4 operon and regulates its expression. Further, we observed that the OmpR-regulated T6SS4 is essential for bacterial survival under acidic conditions and that its expression is induced by low pH. Moreover, we showed that T6SS4 plays a role in pumping H(+) out of the cell to maintain intracellular pH homeostasis. The acid tolerance phenotype of T6SS4 is dependent on the ATPase activity of ClpV4, one of the components of T6SS4. These results not only uncover a novel strategy utilized by Y. pseudotuberculosis for acid resistance, but also reveal that T6SS, a bacteria secretion system known to be functional in protein transportation has an unexpected function in H(+) extrusion under acid conditions.