Flexneri Infection Research Articles

Protein N-myristoylation: functions and mechanisms in control of innate immunity.

Protein N-myristoylation is an important fatty acylation catalyzed by N-myristoyltransferases (NMTs), which are ubiquitous enzymes in eukaryotes. Specifically, attachment of a myristoyl group is vital for proteins participating in various biological functions, including signal transduction, cellular localization, and oncogenesis. Recent studies have revealed unexpected mechanisms indicating that protein N-myristoylation is involved in host defense against microbial and viral infections. In this review, we describe the current understanding of protein N-myristoylation (mainly focusing on myristoyl switches) and summarize its crucial roles in regulating innate immune responses, including TLR4-dependent inflammatory responses and demyristoylation-induced innate immunosuppression during Shigella flexneri infection. Furthermore, we examine the role of myristoylation in viral assembly, intracellular host interactions, and viral spread during human immunodeficiency virus-1 (HIV-1) infection. Deeper insight into the relationship between protein N-myristoylation and innate immunity might enable us to clarify the pathogenesis of certain infectious diseases and better harness protein N-myristoylation for new therapeutics.

MTFMT deficiency correlates with reduced mitochondrial integrity and enhanced host susceptibility to intracellular infection

Mitochondria behave as functional and structural hubs for innate defense against intracellular infection. While the mitochondrial membrane serves as a platform for the assembly of signaling complexes activated by intracellular infection, various danger molecules derived from impaired mitochondria activate innate signaling pathways. Using methionyl-tRNA formyl transferase (MTFMT)-deficient cells, which exhibit impaired mitochondrial activity, we examined the role of mitochondrial integrity in regulating innate defense against infection. Since MTFMT functions at the early steps of mitochondrial translation, its loss was expected to cause defects in mitochondrial activity. Under transient MTFMT gene silencing conditions, we observed shortened mitochondria along with reduced activity. MTFMT-silenced cells were more susceptible to intracellular infection, as examined by infection with RNA viruses and the intracellular bacterium Shigella flexneri. In support of this observation, MTFMT-silenced cells possessed lowered basal NF-κB activity, which remained low after S. flexneri infection. In addition, the mitochondrial accumulation of evolutionarily conserved signaling intermediate in Toll pathway (ECSIT), an adaptor protein for NF-κB activation, was significantly decreased in MTFMT-silenced cells, explaining the reduced NF-κB activity observed in these cells. Since impaired mitochondria likely release mitochondrial molecules, we evaluated the contribution of mitochondrial N-formyl peptides to the regulation of bacterial infection. Transient transfection of mitochondrial-derived N-formyl peptides favored S. flexneri infection, which was accompanied by enhanced bacterial survival, but did not affect host cell viability. However, transient transfection of mitochondrial-derived N-formyl peptides did not affect basal NF-κB activity. Altogether, these data suggest that the integrity of mitochondria is essential to their proper function in protecting against infection, as intact mitochondria not only block the release of danger molecules but also serve as signaling hubs for the downstream NF-κB pathway.

Pathogen-specific risk of seizure in children with moderate-to-severe diarrhoea: Case control study with follow-up.

To determine the pathogen-specific risk of seizure in under-five children hospitalised with moderate-to-severe diarrhoea (MSD) in rural settings. This was a prospective case-control study with follow-up, conducted in a sentinel facility of Global Enteric Multicenter Study in Mirzapur, a rural community of Bangladesh between 2007 and 2010. Children aged 0-59months who presented with MSD and seizure constituted the cases whereas those who did not have seizure comprised the controls. MSD was defined if the episodes were associated with dehydration or dysentery or required hospitalisation with diarrhoea or dysentery. All enrolled children were followed up at home within 50-90days of enrolment. A total of 64 cases and 128 randomly selected controls formed the analysable dataset. The result of logistic regression analysis after adjusting for potential confounders revealed that shigellosis (Shigella species, OR=5.34, 95% CI=2.37-12.04) particularly S. flexneri (OR=3.34, 95% CI=1.48-7.57), S. flexneri 6 (OR=23.24, 95% CI=2.79-193.85), S. sonnei (OR=6.90, 95% CI=2.34-19.85); norovirus (OR=6.77, 95% CI=1.69-27.11), fever (OR=16.75, 95% CI=1.81-154.70) and loss of consciousness (OR=35.25, 95% CI=1.71-726.20) were the independent risk factors for seizure in MSD children. At enrolment, cases had lower WHZ (P=0.006) compared to their peers, follow-up anthropometrics showed significant improvement in WHZ (P<0.001) and WAZ (P<0.05), whereas deterioration in HAZ (P<0.001) in both cases and controls. Childhood MSD episodes particularly due to Shigella and norovirus are often associated with seizure. Prompt identification and appropriate management of children with shigellosis may reduce occurrence and adverse consequences of seizure linked with MSD.

The history of septin biology and bacterial infection

Investigation of cytoskeleton during bacterial infection has significantly contributed to both cell and infection biology. Bacterial pathogens Listeria monocytogenes and Shigella flexneri are widely recognised as paradigms for investigation of the cytoskeleton during bacterial entry, actin-based motility, and cell-autonomous immunity. At the turn of the century, septins were a poorly understood component of the cytoskeleton mostly studied in the context of yeast cell division and human cancer. In 2002, a screen performed in the laboratory of Pascale Cossart identified septin family member MSF (MLL septin-like fusion, now called SEPT9) associated with L. monocytogenes entry into human epithelial cells. These findings inspired the investigation of septins during L. monocytogenes and S. flexneri infection at the Institut Pasteur, illuminating important roles for septins in host-microbe interactions. In this review, we revisit the history of septin biology and bacterial infection, and discuss how the comparative study of L. monocytogenes and S. flexneri has been instrumental to understand septin roles in cellular homeostasis and host defence.

Generating Mucosal and Systemic Immune Response Following Vaccination of Vibrio cholerae Adhesion Molecule against Shigella flexneri Infection

AbstractIntroduction: Previous studies have shown 37.8 kDa pili subunit protein of Vibrio cholerae and 49.8 kDa pili subunit protein of Shigella flexneri can act as an adhesion molecule to initiate infection. These molecules also have the ability to agglutinate blood. The present study assessed mucosal and systemic immunity following vaccination using 37.8 kDa V. cholerae and protection against S. flexneri. Subjects and Methods: Haemagglutination test was performed after purification of V. cholerae protein, followed by an anti-haemagglutination test. The intestinal weight and colony count were used to validate the protective effect on balb/c mice which were divided into the naive group, Shigella-positive control group, Vibrio-positive control group, V. cholerae infected-Vibrio-vaccinated group and S. flexneri-infected-Vibrio-vaccinated group. Th17, Treg, interleukin (IL) IL-17A, β-defensin and secretory-immunoglobulin A (s-IgA) were also measured to determine the systemic and mucosal immunity after vaccination. Results: The haemagglutination and anti-haemagglutination tests showed that the 37.8 kDa protein could inhibit 49.8 kDa of the S. flexneri pili subunit. Decreased intestinal weight and colony count of vaccinated group compared to naive group also support cross reaction findings. Vaccination also generates higher level of Th17, Treg, IL-17A, β-defensin and s-IgA significantly. Conclusions: 37.8 kDa subunit pili can act as a homologous vaccine candidate to prevent V. cholerae and S. flexneri infection.

Reverse vaccinology approach for the identification and characterization of outer membrane proteins of Shigella flexneri as potential cellular- and antibody-dependent vaccine candidates.

PurposeIn the developing world, bacillary dysentery is one of the most common communicable diarrheal infections. There are approximately 169 million cases of shigellosis reported worldwide. The disease is transmitted by a group of Gram-negative intracellular enterobacteria known as Shigella flexneri, S. sonnei, S. dysenteriae, and S. boydii. Conventional treatment regimens for Shigella have been less effective due to the development of resistant strains against antibiotics. Therefore, an effective vaccine for the long term control of Shigella transmission is urgently needed.Materials and MethodsIn this study, a reverse vaccinology approach was employed to identify most conserved and immunogenic outer membrane proteins (OMPs) of S. flexneri 2a.ResultsFive OMPs including fepA, ompC, nlpD_1, tolC, and nlpD_2 were identified as potential vaccine candidates. Protein-protein interactions analysis using STRING software (https://string-db.org/) revealed that five of these OMPs may potentially interact with other intracellular proteins which are involved in beta-lactam resistance pathway. B- and T-cell epitopes of the selected OMPs were predicted using BCPred as well as Propred I and Propred (http://crdd.osdd.net/raghava/propred/), respectively. Each of these OMPs contains regions which are capable to induce B- and T-cell immune responses.ConclusionAnalysis acquired from this study showed that five selected OMPs have great potential for vaccine development against S. flexneri infection. The predicted immunogenic epitopes can also be used for development of peptide vaccines or multi-epitope vaccines against human shigellosis. Reverse vaccinology is a promising strategy for the discovery of potential vaccine candidates which can be used for future vaccine development against global persistent infections.

Mechanisms of bacillary dysentery: lessons learnt from infant rabbits

ABSTRACT The bacterial pathogen Shigella flexneri causes more than 250 million cases of bacillary dysentery (blood in stool) every year across the world. This human-specific disease is characterized by profuse bloody diarrhea, dramatic ulceration of the colonic epithelium and immune cell infiltration of the colonic tissue. A major challenge in understanding the mechanisms supporting bacillary dysentery is the reliance on animal models that do not fully recapitulate the symptoms observed in humans, including bloody diarrhea. Here we outline advances provided by a recently developed infant rabbit model of bacillary dysentery. The infant rabbit model defines bacillary dysentery as a critical combination of massive vascular lesions and dramatic epithelial fenestration due to intracellular infection and cell-to-cell spread, respectively. The infant rabbit model provides an unprecedented framework for understanding how the cell biology of Shigella flexneri infection relates to pathogenesis.

Evaluation of in vitro and in vivo antibiotic efficacy against a novel bioluminescent Shigella flexneri

Shigella spp., the bacteria responsible for shigellosis, are one of the leading causes of diarrheal morbidity and mortality amongst children. There is a pressing need for the development of novel therapeutics, as resistance of Shigella to many currently used antibiotics is rapidly emerging. This paper describes the development of robust in vitro and in vivo tools to study antibiotic efficacy against Shigella flexneri. A novel bioluminescent S. flexneri strain (S. flexneri lux1) was generated, which can be used in a mammalian epithelial cell co-culture assay to evaluate antibiotic intracellular and extracellular efficacy. In addition, the S. flexneri lux1 strain was used with an intraperitoneal (IP) murine model of shigellosis to test the efficacy of ciprofloxacin and ampicillin. Both antibiotics significantly reduced the observed radiance from the gastrointestinal tissue of infected mice compared to vehicle control. Furthermore, plated gastrointestinal tissue homogenate confirmed antibiotic treatment significantly reduced the S. flexneri infection. However, in contrast to the results generated with tissue homogenate, the radiance data was not able to distinguish between the efficacy of ampicillin and ciprofloxacin. Compared to traditional methods, these models can be utilized for efficient screening of novel antibiotics aiding in the discovery of new treatments against shigellosis.

Preferential phosphatidylinositol 5-phosphate binding contributes to a destabilization of the VHS domain structure of Tom1

Tom1 transports endosomal ubiquitinated proteins that are targeted for degradation in the lysosomal pathway. Infection of eukaryotic cells by Shigella flexneri boosts oxygen consumption and promotes the synthesis of phosphatidylinositol-5-phosphate (PtdIns5P), which triggers Tom1 translocation to signaling endosomes. Removing Tom1 from its cargo trafficking function hinders protein degradation in the host and, simultaneously, enables bacterial survival. Tom1 preferentially binds PtdIns5P via its VHS domain, but the effects of a reducing environment as well as PtdIns5P on the domain structure and function are unknown. Thermal denaturation studies demonstrate that, under reducing conditions, the monomeric Tom1 VHS domain switches from a three-state to a two-state transition behavior. PtdIns5P reduced thermostability, interhelical contacts, and conformational compaction of Tom1 VHS, suggesting that the phosphoinositide destabilizes the protein domain. Destabilization of Tom1 VHS structure was also observed with other phospholipids. Isothermal calorimetry data analysis indicates that, unlike ubiquitin, Tom1 VHS endothermically binds to PtdIns5P through two noncooperative binding sites, with its acyl chains playing a relevant role in the interaction. Altogether, these findings provide mechanistic insights about the recognition of PtdIns5P by the VHS domain that may explain how Tom1, when in a different VHS domain conformational state, interacts with downstream effectors under S. flexneri infection.

Shigella promotes major alteration of gut epithelial physiology and tissue invasion by shutting off host intracellular transport

Intracellular trafficking pathways in eukaryotic cells are essential to maintain organelle identity and structure, and to regulate cell communication with its environment. Shigella flexneri invades and subverts the human colonic epithelium by the injection of virulence factors through a type 3 secretion system (T3SS). In this work, we report the multiple effects of two S. flexneri effectors, IpaJ and VirA, which target small GTPases of the Arf and Rab families, consequently inhibiting several intracellular trafficking pathways. IpaJ and VirA induce large-scale impairment of host protein secretion and block the recycling of surface receptors. Moreover, these two effectors decrease clathrin-dependent and -independent endocytosis. Therefore, S. flexneri infection induces a global blockage of host cell intracellular transport, affecting the exchange between cells and their external environment. The combined action of these effectors disorganizes the epithelial cell polarity, disturbs epithelial barrier integrity, promotes multiple invasion events, and enhances the pathogen capacity to penetrate into the colonic tissue in vivo.

The Shigella type three secretion system effector OspF invades host nucleus by binding host importin α1.

Shigella type III effector OspF, a nuclear translocation protein, has been showed to have an essential role in Shigella flexneri infection and virulence; OspF increases inflammation and induces bacterial invasion by down-regulating MAPK kinases pathway. Nevertheless, the exact mechanisms underlying its nuclear translocation and signaling remain unclear. In the following study, we constructed series of OspF truncated mutants to identify the OspF nuclear localization signals which mapped to the amino acids 209-239. Moreover, we found that this sequence was essential to bind the canonical nuclear import adaptor importin α1. Additionally, the 209-239 residues deletion mutant could not bind the importin α1. Consistent with this observation, our results revealed that OspF can inhibit the MAPK signals in the nucleus. Moreover, OspF translocate to the nucleus through Ran-GTPase and importin α1-dependent manner. These results can provide new avenues for depicting the biological functions of OspF during Shigella infection.

The MFS efflux pump EmrKY contributes to the survival of Shigella within macrophages

Efflux pumps are membrane protein complexes conserved in all living organisms. Beyond being involved in antibiotic extrusion in several bacteria, efflux pumps are emerging as relevant players in pathogen-host interactions. We have investigated on the possible role of the efflux pump network in Shigella flexneri, the etiological agent of bacillary dysentery. We have found that S. flexneri has retained 14 of the 20 pumps characterized in Escherichia coli and that their expression is differentially modulated during the intracellular life of Shigella. In particular, the emrKY operon, encoding an efflux pump of the Major Facilitator Superfamily, is specifically and highly induced in Shigella-infected U937 macrophage-like cells and is activated in response to a combination of high K+ and acidic pH, which are sensed by the EvgS/EvgA two-component system. Notably, we show that following S. flexneri infection, macrophage cytosol undergoes a mild reduction of intracellular pH, permitting EvgA to trigger the emrKY activation. Finally, we present data suggesting that EmrKY is required for the survival of Shigella in the harsh macrophage environment, highlighting for the first time the key role of an efflux pump during the Shigella invasive process.

A murine model of diarrhea, growth impairment and metabolic disturbances with Shigella flexneri infection and the role of zinc deficiency

ABSTRACTShigella is one of the major enteric pathogens worldwide. We present a murine model of S. flexneri infection and investigate the role of zinc deficiency (ZD). C57BL/6 mice fed either standard chow (HC) or ZD diets were pretreated with an antibiotic cocktail and received S. flexneri strain 2457T orally. Antibiotic pre-treated ZD mice showed higher S. flexneri colonization than non-treated mice. ZD mice showed persistent colonization for at least 50 days post-infection (pi). S. flexneri-infected mice showed significant weight loss, diarrhea and increased levels of fecal MPO and LCN in both HC and ZD fed mice. S. flexneri preferentially colonized the colon, caused epithelial disruption and inflammatory cell infiltrate, and promoted cytokine production which correlated with weight loss and histopathological changes. Infection with S. flexneri ΔmxiG (critical for type 3 secretion system) did not cause weight loss or diarrhea, and had decreased stool shedding duration and tissue burden. Several biochemical changes related to energy, inflammation and gut-microbial metabolism were observed. Zinc supplementation increased weight gains and reduced intestinal inflammation and stool shedding in ZD infected mice. In conclusion, young antibiotic-treated mice provide a new model of oral S. flexneri infection, with ZD promoting prolonged infection outcomes.

NLRP10 Affects the Stability of Abin-1 To Control Inflammatory Responses.

NOD-like receptors (NLR) are critical regulators of innate immune signaling. The NLR family consists of 22 human proteins with a conserved structure containing a central oligomerization NACHT domain, an N-terminal interaction domain, and a variable number of C-terminal leucine-rich repeats. Most NLR proteins function as cytosolic pattern recognition receptors with activation of downstream inflammasome signaling, NF-κB, or MAPK activation. Although NLRP10 is the only NLR protein lacking the leucine rich repeats, it has been implicated in multiple immune pathways, including the regulation of inflammatory responses toward Leishmania major and Shigella flexneri infection. In this study, we identify Abin-1, a negative regulator of NF-κB, as an interaction partner of NLRP10 that binds to the NACHT domain of NLRP10. Using S. flexneri as an infection model in human epithelial cells, our work reveals a novel function of NLRP10 in destabilizing Abin-1, resulting in enhanced proinflammatory signaling. Our data give insight into the molecular mechanism underlying the function of NLRP10 in innate immune responses.

ADP-heptose is a newly identified pathogen-associated molecular pattern of Shigella flexneri.

During an infection, the detection of pathogens is mediated through the interactions between pathogen-associated molecular patterns (PAMPs) and pathogen recognition receptors. β-Heptose 1,7-bisphosphate (βHBP), an intermediate of the lipopolysaccharide (LPS) biosynthesis pathway, was recently identified as a bacterial PAMP. It was reported that βHBP sensing leads to oligomerization of TIFA proteins, a mechanism controlling NF-κB activation and pro-inflammatory gene expression. Here, we compare the ability of chemically synthesized βHBP and Shigella flexneri lysate to induce TIFA oligomerization in epithelial cells. We find that, unlike bacterial lysate, βHBP fails to initiate rapid TIFA oligomerization. It only induces delayed signaling, suggesting that βHBP must be processed intracellularly to trigger inflammation. Gene deletion and complementation analysis of the LPS biosynthesis pathway revealed that ADP-heptose is the bacterial metabolite responsible for rapid TIFA oligomerization. ADP-heptose sensing occurs down to 10-10M. During S.flexneri infection, it results in cytokine production, a process dependent on the kinase ALPK1. Altogether, our results rule out a major role of βHBP in S.flexneri infection and identify ADP-heptose as a new bacterial PAMP.

Spatial, Temporal, and Functional Assessment of LC3-Dependent Autophagy in Shigella flexneri Dissemination.

Shigella flexneri disseminates within the colonic mucosa by displaying actin-based motility in the cytosol of epithelial cells. Motile bacteria form membrane protrusions that project into adjacent cells and resolve into double-membrane vacuoles (DMVs) from which the bacteria escape, thereby achieving cell-to-cell spread. During dissemination, S. flexneri is targeted by LC3-dependent autophagy, a host cell defense mechanism against intracellular pathogens. The S. flexneri type III secretion system effector protein IcsB was initially proposed to counteract the recruitment of the LC3-dependent autophagy machinery to cytosolic bacteria. However, a recent study proposed that LC3 was recruited to bacteria in DMVs formed during cell-to-cell spread. To resolve the controversy and clarify the role of autophagy in S. flexneri infection, we tracked dissemination using live confocal microscopy and determined the spatial and temporal recruitment of LC3 to bacteria. This approach demonstrated that (i) LC3 was exclusively recruited to wild-type or icsB bacteria located in DMVs and (ii) the icsB mutant was defective in cell-to-cell spread due to failure to escape LC3-positive as well as LC3-negative DMVs. Failure of S. flexneri to escape DMVs correlated with late LC3 recruitment, suggesting that LC3 recruitment is the consequence and not the cause of DMV escape failure. Inhibition of autophagy had no positive impact on the spreading of wild-type or icsB mutant bacteria. Our results unambiguously demonstrate that IcsB is required for DMV escape during cell-to-cell spread, regardless of LC3 recruitment, and do not support the previously proposed notion that autophagy counters S. flexneri dissemination.

Identification of interactions among host and bacterial proteins and evaluation of their role early during Shigella flexneri infection.

Shigella species cause diarrhoea by invading and spreading through the epithelial layer of the human colon. The infection triggers innate immune responses in the host that the bacterium combats by translocating into the host cell cytosol via a type 3 secretion system bacterial effector proteins that interfere with host processes. We previously demonstrated that interaction of the Shigella type 3 secreted effector protein IcsB with the host protein Toca-1 inhibits the innate immune response microtubule-associated protein light-chain 3 (LC3)-associated phagocytosis, and that IcsB interaction with Toca-1 is required for inhibition of this host response. Here, we show that Toca-1 in vitro precipitated not only IcsB, but also the type 3 secreted proteins OspC3, IpgD and IpaB. OspC3 and IpgD precipitation with Toca-1 was dependent on IcsB. Early during infection, most of these proteins localized near intracellular Shigella. We examined whether interactions among these proteins restrict innate host cell responses other than LC3-associated phagocytosis. In infected cells, OspC3 blocks production and secretion of the mature pro-inflammatory cytokine IL-18; however, we found that interaction of OspC3 with IcsB, either directly or indirectly via Toca-1, was not required for OspC3-mediated restriction of IL-18 production. These results indicate that interactions of the host protein Toca-1 with a subset of type 3 effector proteins contribute to the established function of some, but not all involved, effector proteins.

Wiskott-Aldrich syndrome protein regulates autophagy and inflammasome activity in innate immune cells

Dysregulation of autophagy and inflammasome activity contributes to the development of auto-inflammatory diseases. Emerging evidence highlights the importance of the actin cytoskeleton in modulating inflammatory responses. Here we show that deficiency of Wiskott–Aldrich syndrome protein (WASp), which signals to the actin cytoskeleton, modulates autophagy and inflammasome function. In a model of sterile inflammation utilizing TLR4 ligation followed by ATP or nigericin treatment, inflammasome activation is enhanced in monocytes from WAS patients and in WAS-knockout mouse dendritic cells. In ex vivo models of enteropathogenic Escherichia coli and Shigella flexneri infection, WASp deficiency causes defective bacterial clearance, excessive inflammasome activation and host cell death that are associated with dysregulated septin cage-like formation, impaired autophagic p62/LC3 recruitment and defective formation of canonical autophagosomes. Taken together, we propose that dysregulation of autophagy and inflammasome activities contribute to the autoinflammatory manifestations of WAS, thereby identifying potential targets for therapeutic intervention.

Ubiquitination and degradation of GBPs by a Shigella effector to suppress host defence.

Interferon-inducible guanylate-binding proteins (GBPs) mediate cell-autonomous antimicrobial defences. Shigella flexneri, a Gram-negative cytoplasmic free-living bacterium that causes bacillary dysentery, encodes a repertoire of highly similar type III secretion system effectors called invasion plasmid antigen Hs (IpaHs). IpaHs represent a large family of bacterial ubiquitin-ligases, but their function is poorly understood. Here we show that S. flexneri infection induces rapid proteasomal degradation of human guanylate binding protein-1 (hGBP1). We performed a transposon screen to identify a mutation in the S. flexneri gene ipaH9.8 that prevented hGBP1 degradation. IpaH9.8 targets hGBP1 for degradation via Lys48-linked ubiquitination. IpaH9.8 targets multiple GBPs in the cytoplasm independently of their nucleotide-bound states and their differential function in antibacterial defence, promoting S. flexneri replication and resulting in the death of infected mice. In the absence of IpaH9.8, or when binding of GBPs to IpaH9.8 was disrupted, GBPs such as hGBP1 and mouse GBP2 (mGBP2) translocated to intracellular S. flexneri and inhibited bacterial replication. Like wild-type mice, mutant mice deficient in GBP1-3, 5 and 7 succumbed to S. flexneri infection, but unlike wild-type mice, mice deficient in these GBPs were also susceptible to S. flexneri lacking ipaH9.8. The mode of IpaH9.8 action highlights the functional importance of GBPs in antibacterial defences. IpaH9.8 and S. flexneri provide a unique system for dissecting GBP-mediated immunity.

Effective protection of mice against Shigella flexneri with a new self-adjuvant multicomponent vaccine.

The aim of this study was to develop an immunogenic protective product against Shigella flexneri by employing a simple and safe heat treatment-based strategy. The physicochemical characteristics of naturally produced (OMV) and heat-induced (HT) outer-membrane vesicles from S. flexneri were examined, including a comparison of the protein content of the products. Toxicological and biodistribution studies, and a preliminary experiment to examine the protective effectiveness of HT in a murine model of S. flexneri infection, were also included. This method simultaneously achieves complete bacterial inactivation and the production of the HT vaccine product, leading to a safe working process. The obtained HT complex presented a similar morphology (electron microscopy) and chemical composition to the classical OMV, although it was enriched in some immunogens, such as lipoproteins, OmpA or OmpC, among others. The HT formulation was not toxic and biodistribution studies performed in mice demonstrated that the vaccine product remained in the small intestine after nasal administration. Finally, a single dose of HT administered nasally was able to protect mice against S. flexneri 2a. The convenient and safe manufacturing process, and the preliminary biological evaluation, support the use of the self-adjuvanted HT complex as a new vaccine candidate to face shigellosis. Further development is required, such as additional immune analyses, to evaluate whether this new subunit vaccine can be useful in achieving full protection against Shigella.