The Shigella kinase effector OspG modulates host ubiquitin signaling to escape septin-cage entrapment.

Shigella flexneri is a Gram-negative bacterium causing severe bloody dysentery. Its pathogenesis is largely dictated by a plasmid-encoded type III secretion system (T3SS) and its associated effectors. Among these, the effector OspG has been shown to bind to the ubiquitin conjugation machinery (E2~Ub) to activate its kinase activity. However, the cellular targets of OspG remain elusive despite years of extensive efforts. Here we show by unbiased phosphoproteomics that a major target of OspG is CAND1, a regulatory protein controlling the assembly of cullin-RING ubiquitin ligases (CRLs). CAND1 phosphorylation weakens its interaction with cullins, which is expected to impact a large panel of CRL E3s. Indeed, global ubiquitome profiling reveals marked changes in the ubiquitination landscape when OspG is introduced. Notably, OspG promotes ubiquitination of a class of cytoskeletal proteins called septins, thereby inhibiting formation of cage-like structures encircling cytosolic bacteria. Overall, we demonstrate that pathogens have evolved an elaborate strategy to modulate host ubiquitin signaling to evade septin-cage entrapment.

Exploiting bacterial effector proteins to uncover evolutionarily conserved antiviral host machinery.

Arboviruses are a diverse group of insect-transmitted pathogens that pose global public health challenges. Identifying evolutionarily conserved host factors that combat arbovirus replication in disparate eukaryotic hosts is important as they may tip the balance between productive and abortive viral replication, and thus determine virus host range. Here, we exploit naturally abortive arbovirus infections that we identified in lepidopteran cells and use bacterial effector proteins to uncover host factors restricting arbovirus replication. Bacterial effectors are proteins secreted by pathogenic bacteria into eukaryotic hosts cells that can inhibit antimicrobial defenses. Since bacteria and viruses can encounter common host defenses, we hypothesized that some bacterial effectors may inhibit host factors that restrict arbovirus replication in lepidopteran cells. Thus, we used bacterial effectors as molecular tools to identify host factors that restrict four distinct arboviruses in lepidopteran cells. By screening 210 effectors encoded by seven different bacterial pathogens, we identify several effectors that individually rescue the replication of all four arboviruses. We show that these effectors encode diverse enzymatic activities that are required to break arbovirus restriction. We further characterize Shigella flexneri-encoded IpaH4 as an E3 ubiquitin ligase that directly ubiquitinates two evolutionarily conserved proteins, SHOC2 and PSMC1, promoting their degradation in insect and human cells. We show that depletion of either SHOC2 or PSMC1 in insect or human cells promotes arbovirus replication, indicating that these are ancient virus restriction factors conserved across invertebrate and vertebrate hosts. Collectively, our study reveals a novel pathogen-guided approach to identify conserved antimicrobial machinery, new effector functions, and conserved roles for SHOC2 and PSMC1 in virus restriction.

Cascaded signal amplification strategy for ultra-specific, ultra-sensitive, and visual detection of Shigella flexneri.

Pathogen infections including Shigella flexneri have posed a significant threat to human health for numerous years. Although culturing and qPCR were the gold standards for pathogen detection, time-consuming and instrument-dependent restrict their application in rapid diagnosis and economically less-developed regions. Thus, it is urgently needed to develop rapid, simple, sensitive, accurate, and low-cost detection methods for pathogen detection. In this study, an immunomagnetic beads-recombinase polymerase amplification-CRISPR/Cas12a (IMB-RPA-CRISPR/Cas12a) method was built based on a cascaded signal amplification strategy for ultra-specific, ultra-sensitive, and visual detection of S. flexneri in the laboratory. Firstly, S. flexneri was specifically captured and enriched by IMB (Shigella antibody-coated magnetic beads), and the genomic DNA was released and used as the template in the RPA reaction. Then, the RPA products were mixed with the pre-loaded CRISPR/Cas12a for fluorescence visualization. The results were observed by naked eyes under LED blue light, with a sensitivity of 5 CFU/mL in a time of 70 min. With no specialized equipment or complicated technical requirements, the IMB-RPA-CRISPR/Cas12a diagnostic method can be used for visual, rapid, and simple detection of S. flexneri and can be easily adapted to monitoring other pathogens.

Genome-Wide Investigation Reveals Potential Therapeutic Targets in Shigella spp.

Shigella stands as a major contributor to bacterial dysentery worldwide scale, particularly in developing countries with inadequate sanitation and hygiene. The emergence of multidrug-resistant strains exacerbates the challenge of treating Shigella infections, particularly in regions where access to healthcare and alternative antibiotics is limited. Therefore, investigations on how bacteria evade antibiotics and eventually develop resistance could open new avenues for research to develop novel therapeutics. The aim of this study was to analyze whole genome sequence (WGS) of human pathogenic Shigella spp. to elucidate the antibiotic resistance genes (ARGs) and their mechanism of resistance, gene-drug interactions, protein-protein interactions, and functional pathways to screen potential therapeutic candidate(s). We comprehensively analyzed 45 WGS of Shigella, including S. flexneri (n = 17), S. dysenteriae (n = 14), S. boydii (n = 11), and S. sonnei (n = 13), through different bioinformatics tools. Evolutionary phylogenetic analysis showed three distinct clades among the circulating strains of Shigella worldwide, with less genomic diversity. In this study, 2,146 ARGs were predicted in 45 genomes (average 47.69 ARGs/genome), of which only 91 ARGs were found to be shared across the genomes. Majority of these ARGs conferred their resistance through antibiotic efflux pump (51.0%) followed by antibiotic target alteration (23%) and antibiotic target replacement (18%). We identified 13 hub proteins, of which four proteins (e.g., tolC, acrR, mdtA, and gyrA) were detected as potential hub proteins to be associated with antibiotic efflux pump and target alteration mechanisms. These hub proteins were significantly (p < 0.05) enriched in biological process, molecular function, and cellular components. Therefore, the finding of this study suggests that human pathogenic Shigella strains harbored a wide range of ARGs that confer resistance through antibiotic efflux pumps and antibiotic target modification mechanisms, which must be taken into account to devise and formulate treatment strategy against this pathogen. Moreover, the identified hub proteins could be exploited to design and develop novel therapeutics against MDR pathogens like Shigella.

Detection of OXA-181 Carbapenemase in Shigella flexneri.

We report the detection of OXA-181 carbapenemase in an azithromycin-resistant Shigella spp. bacteria in an immunocompromised patient. The emergence of OXA-181 in Shigella spp. bacteria raises concerns about the global dissemination of carbapenem resistance in Enterobacterales and its implications for the treatment of infections caused by Shigella bacteria.

Antimicrobial, anti-adhesive, and anti-invasive effects of condition media derived from adipose mesenchymal stem cells against Shigella flexneri.

The objective of the current study was to examine the antimicrobial, anti-adhesion, and anti-invasion properties of various concentrations of condition media obtained from adipose mesenchymal stem cells (AD-MSCs CM) against Shigella flexneri (S. flexneri). AD-MSCs characterization and antimicrobial assay were performed using flow cytometry and microdilution by colony counting, respectively. For evaluating adhesion and invasion, Caco-2 cells were infected by S. flexneri at three different multiplicities of infection (MOIs of 1, 10, and 50) and then treated with DMEM medium and AD-MSCs CM. The inhibitory effect of AD-MSCs CM was assessed after 24 and 48 h of treatment by CFU (colony-forming unit) counting. A total of 84, 65, and 56% reduction in the adhesion rate of S. flexneri to Caco-2 cells treated with AD-MSCs CM were observed at MOIs of 1, 10, and 50, respectively. While S. flexneri at MOI:1 had no invasive effect on Caco-2 cells, convincing invasion was detected at MOIs of 10 and 50, showing a significant decrease following treatment with AD-MSCs CM. The current study results open new insights into AD-MSCs CM as a new non-antibiotic therapeutic candidate for S. flexneri infections.

Gastrointestinal signals in supplemented media reveal a role in adherence for the Shigella flexneri sap autotransporter gene.

Shigella flexneri causes severe diarrheal disease worldwide. While many aspects of pathogenesis have been elucidated, significant knowledge gaps remain regarding the role of putative chromosomally-encoded virulence genes. The uncharacterized sap gene encoded on the chromosome has significant nucleotide sequence identity to the fluffy (flu) antigen 43 autotransporter gene in pathogenic Escherichia coli. Here, we constructed a Δsap mutant in S. flexneri strain 2457T and examined the effects of this mutation on bacterial cell aggregation, biofilm formation, and adherence to colonic epithelial cells. Analyses included the use of growth media supplemented with glucose and bile salts to replicate small intestinal signals encountered by S. flexneri. Deletion of the sap gene in 2457T affected epithelial cell adherence, resulted in quicker bacterial cell aggregation, but did not affect biofilm formation. This work highlights a functional role for the sap gene in S. flexneri pathogenesis and further demonstrates the importance of using relevant and appropriate gastrointestinal signals to characterize virulence genes of enteropathogenic bacteria.

Shigella induces stress granule formation by ADP-riboxanation of the eIF3 complex.

Under stress conditions, translationally stalled mRNA and associated proteins undergo liquid-liquid phase separation and condense into cytoplasmic foci called stress granules (SGs). Many viruses hijack SGs for their pathogenesis; however, whether pathogenic bacteria also exploit this pathway remains unknown. Here, we report that members of the OspC family of Shigella flexneri induce SG formation in infected cells. Mechanistically, the OspC effectors target multiple subunits of the host translation initiation factor 3 complex by ADP-riboxanation. The modification of eIF3 leads to translational arrest and thus the formation of SGs. Furthermore, OspC-mediated SGs are beneficial for S. flexneri replication within infected host cells, and bacterial strains unable to induce SGs are attenuated for virulence in a murine model of infection. Our findings reveal a mechanism by which bacterial pathogens induce SG assembly by inactivating host translational machinery and promote bacterial proliferation in host cells.

Microtubules provide force to promote membrane uncoating in vacuolar escape for a cyto-invasive bacterial pathogen.

Intracellular bacterial pathogens gain entry to mammalian cells inside a vacuole derived from the host membrane. Some of them escape the bacteria-containing vacuole (BCV) and colonize the cytosol. Bacteria replicating within BCVs coopt the microtubule network to position it within infected cells, whereas the role of microtubules for cyto-invasive pathogens remains obscure. Here, we show that the microtubule motor cytoplasmic dynein-1 and specific activating adaptors are hijacked by the enterobacterium Shigella flexneri. These host proteins were found on infection-associated macropinosomes (IAMs) formed during Shigella internalization. We identified Rab8 and Rab13 as mediators of dynein recruitment and discovered that the Shigella effector protein IpaH7.8 promotes Rab13 retention on moving BCV membrane remnants, thereby facilitating membrane uncoating of the Shigella-containing vacuole. Moreover, the efficient unpeeling of BCV remnants contributes to a successful intercellular spread. Taken together, our work demonstrates how a bacterial pathogen subverts the intracellular transport machinery to secure a cytosolic niche.

PEtN-Modified O-Antigen Enhances Shigella Pathogenesis by Promoting Epithelial Cell Invasion and Inhibiting Complement Binding.

Shigellosis poses an ongoing global public health threat. The presence and length of the O-antigen in lipopolysaccharide play critical roles in Shigella pathogenesis. The plasmid-mediated opt gene encodes a phosphoethanolamine (PEtN) transferase that catalyzes the addition of PEtN to the O-antigen of Shigella flexneri serotype X and Y strains, converting them into serotype Xv and Yv strains, respectively. Since 2002, these modified strains have become prevalent in China. Here we demonstrate that PEtN-mediated O-antigen modification in S. flexneri increase the severity of corneal infection in guinea pigs without any adaptive cost. This heightened virulence is associated with epithelial cell adhesion and invasion, as well as an enhanced inflammatory response of macrophage. Notably, PEtN addition allow S. flexneri to attenuate the binding of complement C3 and better resist phagocytosis, potentially contributing to the retention of S. flexneri in the host environment.

MULTISPECIES SHIGELLA FLEXNERI OUTBREAK IN A ZOOLOGICAL COLLECTION COINCIDING WITH A CLUSTER IN THE LOCAL HUMAN POPULATION.

Shigella flexneri is a nonmotile gram-negative bacillus that affects humans and nonhuman primates. In August 2021, 15 primates at the ABQ BioPark demonstrated clinical signs of Shigella infection: 3 out of 4 Sumatran and hybrid orangutans (Pongo abelii), 6 out of 8 gorillas (Gorilla gorilla), 2 out of 9 chimpanzees (Pan troglodytes), and 4 out of 4 siamangs (Hylobates syndactylus). Three siamangs and one gorilla succumbed to complications of shigellosis during the initial outbreak and a chimpanzee died 10 mon later. Although it is well documented that Shigella may cause morbidity and mortality in nonhuman primates, the rapid and devastating nature of the outbreak, the difference from previous reports in zoological collections (enzootic vs outbreak), and the chronological overlap with the increase in human cases in the region makes discussion of this Shigella outbreak of significance. The cases presented here are significantly different than previous reports, because these were part of an outbreak that arose and subsided, versus other reports where the authors describe an enzootic disease with persistently infected animals. Close communication with the New Mexico Department of Health allowed for the investigation into possible sources of the outbreak, recommendations regarding biosecurity protocols, and staff education.

Shigella generates distinct IAM subpopulations during epithelial cell invasion to promote efficient intracellular niche formation.

The facultative intracellular pathogen Shigella flexneri invades non-phagocytic epithelial gut cells. Through a syringe-like apparatus called type 3 secretion system, it injects effector proteins into the host cell triggering actin rearrangements leading to its uptake within a tight vacuole, termed the bacterial-containing vacuole (BCV). Simultaneously, Shigella induces the formation of large vesicles around the entry site, which we refer to as infection-associated macropinosomes (IAMs). After entry, Shigella ruptures the BCV and escapes into the host cytosol by disassembling the BCV remnants. Previously, IAM formation has been shown to be required for efficient BCV escape, but the molecular events associated with BCV disassembly have remained unclear. To identify host components required for BCV disassembly, we performed a microscopy-based screen to monitor the recruitment of BAR domain-containing proteins, which are a family of host proteins involved in membrane shaping and sensing (e.g. endocytosis and recycling) during Shigella epithelial cell invasion. We identified endosomal recycling BAR protein Sorting Nexin-8 (SNX8) localized to IAMs in a PI(3)P-dependent manner before BCV disassembly. At least two distinct IAM subpopulations around the BCV were found, either being recycled back to cellular compartments such as the plasma membrane or transitioning to become RAB11A positive "contact-IAMs" involved in promoting BCV rupture. The IAM subpopulation duality was marked by the exclusive recruitment of either SNX8 or RAB11A. Hindering PI(3)P production at the IAMs led to an inhibition of SNX8 recruitment at these compartments and delayed both, the step of BCV rupture time and successful BCV disassembly. Finally, siRNA depletion of SNX8 accelerated BCV rupture and unpeeling of BCV remnants, indicating that SNX8 is involved in controlling the timing of the cytosolic release. Overall, our work sheds light on how Shigella establishes its intracellular niche through the subversion of a specific set of IAMs.

Exopolysaccharide secreted by Lactiplantibacillus plantarum Y12 showed inhibitory effect on the pathogenicity of Shigella flexneri in vitro and in vivo.

Shigella flexneri is a prevalent foodborne and waterborne pathogen that threatens human health. Our previous research indicated that the Lactiplantibacillus plantarum Y12 exopolysaccharide (L-EPS) potentially inhibited the pathogenicity of S. flexneri. The in vitro results of this study demonstrated that L-EPS effectively mitigated the symptoms induced by S. flexneri in HT-29 cells, including inhibited gene expression levels of IL-1ß, IL-6, IL-8, TNF-α, TLR 2/4, and NOD1/2; decreased apoptosis ratio; and alleviated damage degree of intestinal barrier function (Zona occludens 1, Occludin, and Claudin-1). The in vivo results demonstrated that S. flexneri treated with L-EPS elicited mild adverse physiological manifestations, an inflammatory response, and tissue damage. The infection of S. flexneri caused significant alterations in the abundance of phylum (Firmicutes, Bacteroidota, Actinobacteriota, and Proteobacteria), family (Lachnospiraceae, Muribaculaceae, Rikenellaceae, Prevotellaceaea, Ruminococcaceae, and Lactobaillaceae), and genus (Escherichia Shigella and Lachnospirillaceae NK4A136 group) within the cecal microbiota. These changes were accompanied by perturbations in taurine and hypotaurine metabolism, tricarboxylic acid (TCA) cycle activity, arginine biosynthesis, and histidine metabolic pathways. However, intervention with L-EPS attenuated the dysbiosis of cecal microbiota and metabolic disturbances. In summary, our research suggested a potential application of L-EPS as a functional food additive for mitigating S. flexneri infection.

Expression and purification of TolC as a recombinant protein vaccine against Shigella flexneri and evaluation of immunogenic response in mice.

BACKGROUND: Shigella is one of the major causes of dysenteric diarrhea, which is known shigelosis. Shigelosis causes 160,000 deaths annually of diarrheal disease in the global scale especially children less than 5 years old. No licensed vaccine is available against shigelosis, therefore, efforts for develop an effective and safe vaccine against Shigella as before needed. The reverse vaccinology (RV) is a novel strategy that evaluate genome or proteome of the organism to find a new promising vaccine candidate. In this study, immunogenicity of a designed-recombinant antigen is evaluated through the in silico studies and animal experiments to predict a new immunogenic candidate against Shigella. METHODS: In the first step, proteome of Shigella flexneri was obtained from UniProtKB and then the outer membrane and extracellular proteins were predicted. In this study TolC as an outer membrane protein was selected and confirmed among candidates. In next steps, pre-selected protein was evaluated for transmembrane domains, homology, conservation, antigenicity, solubility, and B- and T-cell prediction by different online servers. RESULT: TolC as a conserved outer membrane protein, using different immune-informatics tools had acceptable scores and was selected as the immunogenic antigen for animal experiment studies. Recombinant TolC protein after expression and purification, was administered to BALB/c mice over three intraperitoneal routes. The sera of mice was used to evaluate the IgG1 production assay by indirect-ELISA. The immunized mice depicted effective protection against 2LD50 of Shigella. Flexneri ATCC12022 (challenge study). CONCLUSION: Therefore, the reverse vaccinology approach and experimental test results demonstrated that TolC as a novel effective and immunogenic antigen is capable for protection against shigellosis.

The virulence regulator VirB from Shigella flexneri uses a CTP-dependent switch mechanism to activate gene expression.

The transcriptional antisilencer VirB acts as a master regulator of virulence gene expression in the human pathogen Shigella flexneri. It binds DNA sequences (virS) upstream of VirB-dependent promoters and counteracts their silencing by the nucleoid-organizing protein H-NS. However, its precise mode of action remains unclear. Notably, VirB is not a classical transcription factor but related to ParB-type DNA-partitioning proteins, which have recently been recognized as DNA-sliding clamps using CTP binding and hydrolysis to control their DNA entry gate. Here, we show that VirB binds CTP, embraces DNA in a clamp-like fashion upon its CTP-dependent loading at virS sites and slides laterally on DNA after clamp closure. Mutations that prevent CTP-binding block VirB loading in vitro and abolish the formation of VirB nucleoprotein complexes as well as virulence gene expression in vivo. Thus, VirB represents a CTP-dependent molecular switch that uses a loading-and-sliding mechanism to control transcription during bacterial pathogenesis.

(p)ppGpp is required for virulence of Shigella flexneri.

Infection by the enteric pathogen Shigella flexneri requires transit through the gastrointestinal tract and invasion of and replication within the cells of the host colonic epithelium. This process exposes the pathogen to a range of diverse microenvironments. Furthermore, the unique composition and physical environment of the eukaryotic cell cytosol represents a stressful environment for S. flexneri, and extensive physiological adaptations are needed for the bacterium to thrive. In this work, we show that disrupting synthesis of the stringent response alarmone (p)ppGpp in S. flexneri diminished expression of key virulence genes, including ipaA, ipaB, ipaC, and icsA, and it reduced bacterial invasion and intercellular spread. Deletion of the (p)ppGpp synthase gene relA alone had no effect on S. flexneri virulence, but disruption of both relA and the (p)ppGpp synthase/hydrolase gene spoT resulted in loss of (p)ppGpp synthesis and virulence. While the relA spoT deletion mutant was able to invade a cultured human epithelial cell monolayer, albeit at reduced levels, it was unable to maintain the infection and spread to adjacent cells, as indicated by loss of plaque formation. Complementation with spoT on a plasmid vector restored plaque formation. Thus, SpoT alone is sufficient to provide the necessary level of (p)ppGpp for virulence. These results indicate that (p)ppGpp is required for S. flexneri virulence and adaptation to the intracellular environment, adding to the repertoire of signaling pathways that affect Shigella pathogenesis.

Molecular mechanism of Hfq-dependent sRNA1039 and sRNA1600 regulating antibiotic resistance and virulence in Shigella sonnei.

Bacillary dysentery caused by Shigella spp. is a significant concern for human health. Small non-coding RNA (sRNA) plays a crucial role in regulating antibiotic resistance and virulence in Shigella spp. However, the specific mechanisms behind this phenomenon are still not fully understood. This study discovered two sRNAs (sRNA1039 and sRNA1600) that may be involved in bacterial resistance and virulence. By constructing deletion mutants (WT/ΔSR1039 and WT/ΔSR1600), this study found that the WT/ΔSR1039 mutants caused a two-fold increase in sensitivity to ampicillin, gentamicin and cefuroxime, and the WT/ΔSR1600 mutants caused a two-fold increase in sensitivity to cefuroxime. Furthermore, the WT/ΔSR1600 mutants caused a decrease in the adhesion and invasion of bacteria to HeLa cells (P<0.01), and changed the oxidative stress level of bacteria to reduce their survival rate (P<0.001). Subsequently, this study explored the molecular mechanisms by which sRNA1039 and sRNA1600 regulate antibiotic resistance and virulence. The deletion of sRNA1039 accelerated the degradation of target gene cfa mRNA and reduced its expression, thereby regulating the expression of pore protein gene ompD indirectly and negatively to increase bacterial sensitivity to ampicillin, gentamicin and cefuroxime. The inactivation of sRNA1600 reduced the formation of persister cells to reduce resistance to cefuroxime, and reduced the expression of type-III-secretion-system-related genes to reduce bacterial virulence by reducing the expression of target gene tomB. These results provide new insights into Hfq-sRNA-mRNA regulation of the resistance and virulence network of Shigella sonnei, which could potentially promote the development of more effective treatment strategies.

Transcriptional profiling of zebrafish identifies host factors controlling susceptibility to Shigella flexneri.

Shigella flexneri is a human-adapted pathovar of Escherichia coli that can invade the intestinal epithelium, causing inflammation and bacillary dysentery. Although an important human pathogen, the host response to S. flexneri has not been fully described. Zebrafish larvae represent a valuable model for studying human infections in vivo. Here, we use a Shigella-zebrafish infection model to generate mRNA expression profiles of host response to Shigella infection at the whole-animal level. Immune response-related processes dominate the signature of early Shigella infection (6 h post-infection). Consistent with its clearance from the host, the signature of late Shigella infection (24 h post-infection) is significantly changed, and only a small set of immune-related genes remain differentially expressed, including acod1 and gpr84. Using mutant lines generated by ENU, CRISPR mutagenesis and F0 crispants, we show that acod1- and gpr84-deficient larvae are more susceptible to Shigella infection. Together, these results highlight the power of zebrafish to model infection by bacterial pathogens and reveal the mRNA expression of the early (acutely infected) and late (clearing) host response to Shigella infection.

Refining Immunogenicity through Intradermal Delivery of Outer Membrane Vesicles against Shigella flexneri in Mice.

Shigellosis remains a global health concern, especially in low- and middle-income countries. Despite improvements in sanitation, the absence of a licensed vaccine for human use has prompted global health organizations to support the development of a safe and effective multivalent vaccine that is cost-effective and accessible for limited-resource regions. Outer Membrane Vesicles (OMVs) have emerged in recent years as an alternative to live attenuated or whole-inactivated vaccines due to their immunogenicity and self-adjuvating properties. Previous works have demonstrated the safety and protective capacity of OMVs against Shigella flexneri infection in mouse models when administered through mucosal or intradermal routes. However, some immunological properties, such as the cellular response or cross-protection among different Shigella strains, remained unexplored. In this study, we demonstrate that intradermal immunization of OMVs with needle-free devices recruits a high number of immune cells in the dermis, leading to a robust cellular response marked by antigen-specific cytokine release and activation of effector CD4 T cells. Additionally, functional antibodies are generated, neutralizing various Shigella serotypes, suggesting cross-protective capacity. These findings highlight the potential of OMVs as a promising vaccine platform against shigellosis and support intradermal administration as a simple and painless vaccination strategy to address this health challenge.

Bactericidal effect of low-temperature atmospheric plasma against the Shigella flexneri.

BACKGROUND: Shigella flexneri (S. flexneri) is a common intestinal pathogenic bacteria that mainly causes bacillary dysentery, especially in low socioeconomic countries. This study aimed to apply cold atmospheric plasma (CAP) on S. flexneri directly to achieve rapid, efficient and environmentally friendly sterilization. METHODS: The operating parameters of the equipment were determined by plasma diagnostics. The plate count and transmission electron microscope were employed to calculate bacterial mortality rates and observe the morphological damage of bacterial cells. Measurement of intracellular reactive oxygen species (ROS) and superoxide anions were detected by 2,7-dichlorodihydrofluorescein (DCFH) and Dihydroethidium fluorescence probes, respectively. The fluorescence intensity (a. u.) reflects the relative contents. Additionally, the experiment about the single effect of temperature, ultraviolet (UV), and ROS on bacteria was conducted. RESULTS: The peak discharge voltage and current during plasma operation were 3.92kV and 66mA. After discharge, the bacterial mortality rate of 10, 20, 30 and 40 s of plasma treatment was 60.71%, 74.02%, 88.11% and 98.76%, respectively. It was shown that the intracellular ROS content was proportional to the plasma treatment time and ROS was the major contributor to bacterial death. CONCLUSION: In summary, our results illustrated that the plasma treatment could inactivate S. flexneri efficiently, and the ROS produced by plasma is the leading cause of bacterial mortality. This highly efficient sterilization method renders plasma a highly promising solution for hospitals, clinics, and daily life.