Shigella Virulence Research Articles

Shigella Senses the Environmental Cue Leucine to Promote its Virulence Gene Expression in the Colon

Shigella is a foodborne enteropathogenic bacteria that causes severe bacillary dysentery in humans. Shigella primarily colonizes the human colon and causes disease via invasion of colon epithelial cells. However, the signal regulatory mechanisms associated with its colonization and pathogenesis in the colon remain poorly defined. Here, we report a leucine-mediated regulatory mechanism that promotes Shigella virulence gene expression and invasion of colon epithelial cells. Shigella in response to leucine, which is highly abundant in the colon, via the leucine-responsive regulator Lrp and the binding of Lrp with leucine induces the expression of a newly identified small RNA SsrV. SsrV then activates the expression of virF and downstream invasion-related virulence genes by increasing the protein level of the LysR-type transcription regulator LrhA, therefore enabling Shigella invasion of colon epithelial cells. Shigella lacking ssrV displays impaired invasion ability. Collectively, these findings suggest that Shigella employs a leucine-responsive environmental activation mechanism to establish colonization and pathogenicity.

Mapping the functional B-cell epitopes of Shigella invasion plasmid antigen D (IpaD).

Shigella bacteria utilize the type III secretion system (T3SS) to invade host cells and establish local infection. Invasion plasmid antigen D (IpaD), a component of Shigella T3SS, has garnered extensive interest as a vaccine target, primarily due to its pivotal role in the Shigella invasion, immunogenic property, and a high degree of conservation across Shigella species and serotypes. Currently, we are developing an epitope- and structure-based multivalent vaccine against shigellosis and require functional epitope antigens of key Shigella virulence determinants including IpaD. However, individual IpaD B-cell epitopes, their contributions to the overall immunogenicity, and functional activities attributing to bacteria invasion have not been fully characterized. In this study, we predicted continuous B-cell epitopes in silico and fused each epitope to a carrier protein. Then, we immunized mice intramuscularly with each epitope fusion protein, examined the IpaD-specific antibody responses, and measured antibodies from each epitope fusion for the activity against Shigella invasion in vitro. Data showed that all epitope fusion proteins induced similar levels of anti-IpaD IgG antibodies in mice, and differences were noted for antibody inhibition activity against Shigella invasion. IpaD epitope 1 (SPGGNDGNSV), IpaD epitope 2 (LGGNGEVVLDNA), and IpaD epitope 5 (SPNNTNGSSTET) induced antibodies significantly better in inhibiting invasion from Shigella flexneri 2a, and epitopes 1 and 5 elicited antibodies more effectively at preventing invasion of Shigella sonnei. These results suggest that IpaD epitopes 1 and 5 can be the IpaD representative antigens for epitope-based polyvalent protein construction and protein-based cross-protective Shigella vaccine development.IMPORTANCEShigella is a leading cause of diarrhea in children younger than 5 years in developing countries (children's diarrhea) and continues to be a major threat to public health. No licensed vaccines are currently available against the heterogeneous Shigella species and serotype strains. Aiming to develop a cross-protective multivalent vaccine against shigellosis and dysentery, we applied novel multiepitope fusion antigen (MEFA) technology to construct a broadly immunogenic polyvalent protein antigen, by presenting functional epitopes of multiple Shigella virulence determinants on a backbone protein. The functional IpaD epitopes identified from this study will essentially allow us to construct an optimal polyvalent Shigella immunogen, leading to the development of a cross-protective vaccine against shigellosis (and dysentery) and the improvement of global health. In addition, identifying functional epitopes from heterogeneous virulence determinants and using them as antigenic representatives for the development of cross-protective multivalent vaccines can be applied generally in vaccine development.

Structural and functional characterization of the IpaD π-helix reveals critical roles in DOC interaction, T3SS apparatus maturation, and Shigella virulence

Shigella spp. are highly pathogenic members of the Enterobacteriaceae family, causing ∼269 million cases of bacillary dysentery and >200,000 deaths each year. Like many Gram-negative pathogens, Shigella rely on their type three secretion system (T3SS) to inject effector proteins into eukaryotic host cells, driving both cellular invasion and evasion of host immune responses. Exposure to the bile salt deoxycholate (DOC) significantly enhances Shigella virulence and is proposed to serve as a critical environmental signal present in the small intestine that prepares Shigella’s T3SS for efficient infection of the colonic epithelium. Here, we uncover critical mechanistic details of the Shigella-specific DOC signaling process by describing the role of a π-helix secondary structure element within the T3SS tip protein IpaD. Biophysical characterization and high-resolution structures of IpaD mutants lacking the π-helix show that it is not required for global protein structure, but that it defines the native DOC binding site and prevents off target interactions. Additionally, Shigella strains expressing the π-helix deletion mutants illustrate the pathogenic importance of its role in guiding DOC interaction as flow cytometry and gentamycin protection assays show that the IpaD π-helix is essential for DOC-mediated apparatus maturation and enhanced invasion of eukaryotic cells. Together, these findings add to our understanding of the complex Shigella pathogenesis pathway and its evolution to respond to environmental bile salts by identifying the π-helix in IpaD as a critical structural element required for translating DOC exposure to virulence enhancement.

Production of egg yolk antibody (IgY) against a chimeric protein containing IpaD, StxB, and TolC antigens from Shigella: An investigation of its prophylactic effects against Shiga toxin (Stx) and Shigella dysenteriae in vitro and in vivo

Shigella is a major problem in developing countries. Immunoglobulin Y (IgY) can be used for prophylaxis and neutralize bacteria. The aim of this study was to produce IgY against the chimeric protein containing IpaD, StxB, and TolC antigens from Shigella, investigate its prophylactic and neutralizing effects against Stx and Shigella dysenteriae. The nucleotide sequence corresponding to the chimeric protein was cloned into pET28a plasmid and expressed in E. coli BL21 (DE3). Protein expression was confirmed by SDS-PAGE and the recombinant protein was purified by Ni-NTA affinity chromatography. The 150 μg of chimeric protein was mixed with Freund's adjutant and injected into laying hens (Leghorn). IgY was purified using PEG6000 precipitation. Antibody titer in the serum and egg yolk was evaluated by ELISA. IgY challenge against 1,10 and 50 LD50 of Stx and S. dysenteriae was investigated. A 60.6 kDa recombinant protein was confirmed by SDS-PAGE. ELISA showed that the antibody titer was significantly increased. MTT assay [3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide] showed that at 16 μmol/L, IgY protected HeLa cells against Stx. Treatment of mice with 1000 and 1500 μg IgY leads to complete survival of the mice against 1LD50 toxin and 4000 μg of IgY led to complete survival against 1LD50, also 70% and 30% survival against 10 and 50 LD50S. dysenteriae. This study showed that IgY produced against Stx and Shigella virulence factors could cause high protective effects against bacteria and toxins.

Shigella virulence protein VirG is a broadly protective antigen and vaccine candidate

Diarrhea caused by Shigella has been associated with high morbidity and mortality in young children worldwide. There are no licensed vaccines, and those clinically advanced have restricted coverage as they elicit serotype-specific immunity while disease is caused by multiple circulating serotypes. Our group had previously reported a close association between serum antibodies to the Shigella virulence factor VirG (or IcsA) and clinical protection in infected individuals. VirG is highly conserved among Shigella strains and appealing as a broad-spectrum vaccine candidate. In this study, we investigated the immunogenicity and protective capacity of VirG as a subunit vaccine in mice. The surface-exposed alpha (α) domain of VirG (VirGα) was produced as a recombinant protein. This region has almost identical immune reactivity to full-length VirG. Administered intramuscularly with alum, VirGα elicited robust immune responses and high protective efficacy against S. flexneri 2a and S. sonnei. Almost complete protection was afforded by VirGα given intranasally with the E. coli double mutant heat-labile toxin (dmLT). VirGα-specific antibodies recognized VirG expressed on live Shigella, and blocked Shigella adhesion and invasion to human colonic cells. These results show for the first time that VirGα is a promising cross-protective vaccine candidate to prevent Shigella infection.

RACK1 promotes Shigella flexneri actin-mediated invasion, motility, and cell-to-cell spreading

Shigella flexneri is an intracellular bacterium that hijacks the host actin cytoskeleton to invade and disseminate within the colonic epithelium. Shigella's virulence factors induce actin polymerization, leading to bacterial uptake, actin tail formation, actin-mediated motility, and cell-to-cell spreading. Many host factors involved in the Shigella-prompted actin rearrangements remain elusive. Here, we studied the role of a host protein receptor for activated C kinase 1 (RACK1) in actin cytoskeleton dynamics and Shigella infection. We used time-lapse imaging to demonstrate that RACK1 facilitates Shigella-induced actin cytoskeleton remodeling at multiple levels during infection of epithelial cells. Silencing RACK1 expression impaired Shigella-induced rapid polymerizing structures, reducing host cell invasion, bacterial motility, and cell-to-cell spreading. In uninfected cells, RACK1 silencing reduced jasplakinolide-mediated filamentous actin aggregate formation and negatively affected actin turnover in fast polymerizing structures, such as membrane ruffles. Our findings provide a role of RACK1 in actin cytoskeleton dynamics and Shigella infection.

Genomic, transcriptomic, and phenotypic differences among archetype Shigella flexneri strains of serotypes 2a, 3a, and 6.

Given the genomic diversity between S. flexneri serotypes and the paucity of data to support serotype-specific phenotypic differences, we applied in silico and in vitro functional analyses of archetype strains of 2457T (Sf2a), J17B (Sf3a), and CH060 (Sf6). These archetype strains represent the three leading S. flexneri serotypes recommended for inclusion in multivalent vaccines. Characterizing the genomic and phenotypic variation among these clinically prevalent serotypes is an important step toward understanding serotype-specific host-pathogen interactions to optimize the efficacy of multivalent vaccines and therapeutics. This study underpins the importance for further large-scale serotype-targeted analyses.

VirB, a key transcriptional regulator of Shigella virulence, requires a CTP ligand for its regulatory activities.

Shigella species cause bacillary dysentery, the second leading cause of diarrheal deaths worldwide. There is a pressing need to identify novel molecular drug targets. Shigella virulence phenotypes are controlled by the transcriptional regulator, VirB. We show that VirB belongs to a fast-evolving, plasmid-borne clade of the ParB superfamily, which has diverged from versions with a distinct cellular role-DNA partitioning. We report that, like classic members of the ParB family, VirB binds a highly unusual ligand, CTP. Mutants predicted to be defective in CTP binding are compromised in a variety of virulence attributes controlled by VirB, likely because these mutants cannot engage DNA. This study (i) reveals that VirB binds CTP, (ii) provides a link between VirB-CTP interactions and Shigella virulence phenotypes, (iii) provides new insight into VirB-CTP-DNA interactions, and (iv) broadens our understanding of the ParB superfamily, a group of bacterial proteins that play critical roles in many bacteria.

Diffusible signal factors (DSFs) bind and repress VirF, the leading virulence activator of Shigella flexneri

Shigella, the aetiological agent of human bacillary dysentery, controls the expression of its virulence determinants through an environmentally stimulated cascade of transcriptional activators. VirF is the leading activator and is essential for proper virulence expression. In this work, we report on in vitro and in vivo experiments showing that two autoinducers of the DSF family, XcDSF and BDSF interact with the jelly roll module of VirF causing its inhibition and affecting the expression of the entire virulence system of Shigella, including its ability to invade epithelial cells. We propose a molecular model explaining how the binding of XcDSF and BDSF causes inhibition of VirF by preventing its dimerization. Overall, our experimental results suggest that XcDSF and BDSF may contribute to ”colonisation resistance” in the human gut or, alternatively, may be exploited for the fine-tuning of Shigella virulence expression as the bacterium migrates from the lumen to approach the intestinal mucosa. Our findings also stress how a detailed understanding of the interaction of DSF ligands with VirF may contribute to the rational development of innovative antivirulence drugs to treat shigellosis.

Novel inhibitors that target bacterial virulence identified via HTS against intra-macrophage survival of Shigella flexneri.

Shigella flexneri is a facultative intracellular pathogen that causes shigellosis, a human diarrheal disease characterized by the destruction of the colonic epithelium. Novel antimicrobial compounds to treat infections are urgently needed due to the proliferation of bacterial antibiotic resistance and lack of new effective antimicrobials in the market. Our approach to find compounds that block the Shigella virulence pathway has three potential advantages: (i) resistance development should be minimized due to the lack of growth selection pressure, (ii) no resistance due to environmental antibiotic exposure should be developed since the virulence pathways are not activated outside of host infection, and (iii) the normal intestinal microbiota, which do not have the targeted virulence pathways, should be unharmed. We chose to utilize two phenotypic assays, inhibition of Shigella survival in macrophages and Shigella growth inhibition (minimum inhibitory concentration), to interrogate the 1.7 M compound screening collection subset of the GlaxoSmithKline drug discovery chemical library. A number of secondary assays on the hit compounds resulting from the primary screens were conducted, which, in combination with chemical, structural, and physical property analyses, narrowed the final hit list to 44 promising compounds for further drug discovery efforts. IMPORTANCE The rapid development of antibiotic resistance is a critical problem that has the potential of returning the world to a "pre-antibiotic" type of environment, where millions of people will die from previously treatable infections. One relatively newer approach to minimize the selection pressures for the development of resistance is to target virulence pathways. This is anticipated to eliminate any resistance selection pressure in environmental exposure to virulence-targeted antibiotics and will have the added benefit of not affecting the non-virulent microbiome. This paper describes the development and application of a simple, reproducible, and sensitive assay to interrogate an extensive chemical library in high-throughput screening format for activity against the survival of Shigella flexneri 2457T-nl in THP-1 macrophages. The ability to screen very large numbers of compounds in a reasonable time frame (~1.7 M compounds in ~8 months) distinguishes this assay as a powerful tool in further exploring new compounds with intracellular effect on S. flexneri or other pathogens with similar pathways of pathogenesis. The assay utilizes a luciferase reporter which is extremely rapid, simple, relatively inexpensive, and sensitive and possesses a broad linear range. The assay also utilized THP-1 cells that resemble primary monocytes and macrophages in morphology and differentiation properties. THP-1 cells have advantages over human primary monocytes or macrophages because they are highly plastic and their homogeneous genetic background minimizes the degree of variability in the cell phenotype (1). The intracellular and virulence-targeted selectivity of our methodology, determined via secondary screening, is an enormous advantage. Our main interest focuses on hits that are targeting virulence, and the most promising compounds with adequate physicochemical and drug metabolism and pharmacokinetic (DMPK) properties will be progressed to a suitable in vivo shigellosis model to evaluate the therapeutic potential of this approach. Additionally, compounds that act via a host-directed mechanism could be a promising source for further research given that it would allow a whole new, specific, and controlled approach to the treatment of diseases caused by some pathogenic bacteria.

Differential regulation of Shigella Spa47 ATPase activity by a native C-terminal product of Spa33.

Shigella is a Gram-negative bacterial pathogen that relies on a single type three secretion system (T3SS) as its primary virulence factor. The T3SS includes a highly conserved needle-like apparatus that directly injects bacterial effector proteins into host cells, subverting host cell function, initiating infection, and circumventing resulting host immune responses. Recent findings have located the T3SS ATPase Spa47 to the base of the Shigella T3SS apparatus and have correlated its catalytic function to apparatus formation, protein effector secretion, and overall pathogen virulence. This critical correlation makes Spa47 ATPase activity regulation a likely point of native control over Shigella virulence and a high interest target for non-antibiotic- based therapeutics. Here, we provide a detailed characterization of the natural 11.6 kDa C-terminal translation product of the Shigella T3SS protein Spa33 (Spa33C), showing that it is required for proper virulence and that it pulls down with several known T3SS proteins, consistent with a structural role within the sorting platform of the T3SS apparatus. In vitro binding assays and detailed kinetic analyses suggest an additional role, however, as Spa33C differentially regulates Spa47 ATPase activity based on Spa47s oligomeric state, downregulating Spa47 monomer activity and upregulating activity of both homo-oligomeric Spa47 and the hetero-oligomeric MxiN2Spa47 complex. These findings identify Spa33C as only the second known differential T3SS ATPase regulator to date, with the Shigella protein MxiN representing the other. Describing this differential regulatory protein pair begins to close an important gap in understanding of how Shigella may modulate virulence through Spa47 activity and T3SS function.

Fatty Acids Abolish Shigella Virulence by Inhibiting Its Master Regulator, VirF.

The pathogenicity of Shigella, the intracellular pathogen responsible for human bacillary dysentery, depends on a coordinated and tightly regulated expression of its virulence determinants. This is the result of a cascade organization of its positive regulators, with VirF, a transcriptional activator belonging to the AraC-XylS family, in a pivotal position. VirF itself is submitted to several well-known regulations at the transcriptional level. In this work, we present evidence for a novel posttranslational regulatory mechanism of VirF mediated by the inhibitory interaction with specific fatty acids. By homology modeling and molecular docking analyses, we identify a jelly roll motif in the structure of ViF capable of interacting with medium-chain saturated and long-chain unsaturated fatty acids. In vitro and in vivo assays show that capric, lauric, myristoleic, palmitoleic, and sapienic acids interact effectively with the VirF protein, abolishing its transcription-promoting activity. This silences the virulence system of Shigella, leading to a drastic reduction in its ability to invade epithelial cells and proliferate in their cytoplasm. IMPORTANCE In the absence of a valid vaccine, the main therapeutic approach currently used to treat shigellosis is based on the use of antibiotics. The emergence of antibiotic resistance jeopardizes the future effectiveness of this approach. The importance of the present work resides both in the identification of a new level of posttranslational regulation of the Shigella virulence system and in the characterization of a mechanism offering new opportunities for the design of antivirulence compounds, which may change the treatment paradigm of Shigella infections by limiting the emergence of antibiotic-resistant bacteria.

Formate Metabolism in Shigella flexneri and Its Effect on HeLa Cells at Different Stages during the Infectious Process.

Shigellosis caused by Shigella is one of the most important foodborne illnesses in global health, but little is known about the metabolic cross talk between this bacterial pathogen and its host cells during the different stages of the infection process. A detailed understanding of the metabolism can potentially lead to new drug targets remedying the pressing problem of antibiotic resistance. Here, we use stable isotope-resolved metabolomics as an unbiased and fast method to investigate how Shigella metabolizes 13C-glucose in three different environments: inside the host cells, adhering to the host cells, and alone in suspension. We find that especially formate metabolism by bacteria is sensitive to these different environments. The role of formate in pathogen metabolism is sparsely described in the literature compared to the roles of acetate and butyrate. However, its metabolic pathway is regarded as a potential drug target due to its production in microorganisms and its absence in humans. Our study provides new knowledge about the regulatory effect of formate. Bacterial metabolism of formate is pH dependent when studied alone in culture medium, whereas this effect is less pronounced when the bacteria adhere to the host cells. Once the bacteria are inside the host cells, we find that formate accumulation is reduced. Formate also affects the host cells resulting in a reduced infection rate. This was correlated to an increased immune response. Thus, intriguingly formate plays a double role in pathogenesis by increasing the virulence of Shigella and at the same time stimulating the immune response of the host. IMPORTANCE Bacterial infection is a pressing societal concern due to development of resistance toward known antibiotics. Central carbon metabolism has been suggested as a potential new target for drug development, but metabolic changes upon infection remain incompletely understood. Here, we used a cellular infection model to study how the bacterial pathogen Shigella adapts its metabolism depending on the environment starting from the extracellular medium until Shigella successfully invaded and proliferated inside host cells. The mixed-acid fermentation of Shigella was the major metabolic pathway during the infectious process, and the glucose-derived metabolite formate surprisingly played a divergent role in the pathogen and in the host cell. Our data show reduced infection rate when both host cells and bacteria were treated with formate, which correlated with an upregulated immune response in the host cells. The formate metabolism in Shigella thus potentially provides a route toward alternative treatment strategies for Shigella prevention.

Localized modulation of DNA supercoiling, triggered by the Shigella anti-silencer VirB, is sufficient to relieve H-NS-mediated silencing.

In Bacteria, nucleoid structuring proteins govern nucleoid dynamics and regulate transcription. In Shigella spp., at ≤30°C, the histone-like nucleoid structuring protein (H-NS) transcriptionally silences many genes on the large virulence plasmid. Upon a switch to 37°C, VirB, a DNA binding protein and key transcriptional regulator of Shigella virulence, is produced. VirB functions to counter H-NS-mediated silencing in a process called transcriptional anti-silencing. Here, we show that VirB mediates a loss of negative DNA supercoils from our plasmid-borne, VirB-regulated PicsP-lacZ reporterin vivo. The changes are not caused by a VirB-dependent increase in transcription, nor do they require the presence of H-NS. Instead, the VirB-dependent change in DNA supercoiling requires the interaction of VirB with its DNA binding site, a critical first step in VirB-dependent gene regulation. Using two complementary approaches, we show that VirB:DNA interactions in vitro introduce positive supercoils in plasmid DNA. Subsequently, by exploiting transcription-coupled DNA supercoiling, we reveal that a localized loss of negative supercoils is sufficient to alleviate H-NS-mediated transcriptional silencingindependently of VirB. Together, our findings provide novel insight into VirB,a central regulator of Shigella virulenceand, more broadly, a molecular mechanism that offsets H-NS-dependent silencing of transcription in bacteria.

Evaluation of the impact of Shigella virulence genes on the basis of clinical features observed in patients with shigellosis.

Shigella continues to cause significant morbidity and mortality each year, mostly in under-five children living in developing countries. We investigated the association between Shigella virulence genes and shigellosis. We randomly selected 61 S. flexneri strains isolated from patients in Bangladesh between 2009 and 2013, and evaluated the presence of 140 MDa large-virulence-plasmid (p140), and 22 virulence genes including ipaH, ial, toxin, and T3SS-related genes. We found p140 in 79% (n = 48) and ipaBCD in 90% (n = 55) strains, while seven strains were missing the p140. The prevalence of ial was 89%, ipgC and ipgE was 85%, and the prevalence for the remaining genes was < 85%. During the multivariate analysis, we found that instead of sen, the Shigella enterotoxin gene set along with several other virulence genes such as ipgA, icsB, ipgB1, spa15, and mxiC, were significantly influencing multiple clinical features relevant to shigellosis, including bloody stool, mucoid stool, and rectal straining. We believe our model will help to determine the actual disease burden by directly looking for the genetic material in clinically suggestive patients, especially when detecting the causative organisms by traditional means is difficult.

Roles of Two-Component Signal Transduction Systems in Shigella Virulence

Two-component signal transduction systems (TCSs) are widespread types of protein machinery, typically consisting of a histidine kinase membrane sensor and a cytoplasmic transcriptional regulator that can sense and respond to environmental signals. TCSs are responsible for modulating genes involved in a multitude of bacterial functions, including cell division, motility, differentiation, biofilm formation, antibiotic resistance, and virulence. Pathogenic bacteria exploit the capabilities of TCSs to reprogram gene expression according to the different niches they encounter during host infection. This review focuses on the role of TCSs in regulating the virulence phenotype of Shigella, an intracellular pathogen responsible for severe human enteric syndrome. The pathogenicity of Shigella is the result of the complex action of a wide number of virulence determinants located on the chromosome and on a large virulence plasmid. In particular, we will discuss how five TCSs, EnvZ/OmpR, CpxA/CpxR, ArcB/ArcA, PhoQ/PhoP, and EvgS/EvgA, contribute to linking environmental stimuli to the expression of genes related to virulence and fitness within the host. Considering the relevance of TCSs in the expression of virulence in pathogenic bacteria, the identification of drugs that inhibit TCS function may represent a promising approach to combat bacterial infections.

Golgi stress induces SIRT2 to counteract Shigella infection via defatty-acylation

Enzymes from pathogens often modulate host protein post-translational modifications (PTMs), facilitating survival and proliferation of pathogens. Shigella virulence factors IpaJ and IcsB induce proteolytic cleavage and lysine fatty acylation on host proteins, which cause Golgi stress and suppress innate immunity, respectively. However, it is unknown whether host enzymes could reverse such modifications introduced by pathogens’ virulence factors to suppress pathogenesis. Herein, we report that SIRT2, a potent lysine defatty-acylase, is upregulated by the transcription factor CREB3 under Golgi stress induced by Shigella infection. SIRT2 in turn removes the lysine fatty acylation introduced by Shigella virulence factor IcsB to enhance host innate immunity. SIRT2 knockout mice are more susceptible to Shigella infection than wildtype mice, demonstrating the importance of SIRT2 to counteract Shigella infection.

YfiB: An Outer Membrane Protein Involved in the Virulence of Shigella flexneri.

The intracellular pathogen Shigella flexneri, which is the causative agent of bacillary dysentery, significantly influences the worldwide implication of diarrheal infections, consequentially causing about 1.1 million deaths each year. Due to a nonavailability of an authorized vaccine and the upsurge of multidrug resistance amongst Shigella strains, there has been a huge demand for further genetic analyses which could help in the advancement of new/improved drugs, and finding vaccine candidates against the pathogen. The present study aims to illustrate the role of the yfiB gene in Shigella virulence, part of the periplasmic YfiBNR tripartite signalling system. This system is involved in the regulation of cyclic-di-GMP levels inside the bacterial cells, a vital messenger molecule impacting varied cellular processes such as biofilm formation, cytotoxicity, motility, synthesis of exopolysaccharide, and other virulence mechanisms such as adhesion and invasion of the bacteria. Through a combination of genetic, biochemical, and virulence assays, we show how knocking out the yfiB gene can disrupt the entire YfiBNR system and affect the native c-di-GMP levels. We found that this subsequently causes a negative effect on the biofilm formation, bacterial invasion, host–surface attachment, and the overall virulence of Shigella. This study also carried out a structural and functional assessment of the YfiB protein and determined critical amino acid residues, essential for proper functioning of this signalling system. The present work improves our understanding of the in vivo persistence and survival of Shigella, brings light to the c-di-GMP led regulation of Shigella virulence, and provides a prospective new target to design anti-infection drugs and vaccines against S. flexneri and other bacterial pathogens.

Phosphomimetic Tyrosine Mutations in Spa47 Inhibit Type Three Secretion ATPase Activity and Shigella Virulence Phenotype.

Shigella is a highly infectious human pathogen responsible for 269 million infections and 200,000 deaths per year. Shigella virulence is absolutely reliant on the injection of effector proteins into the host cell cytoplasm via its type three secretion system (T3SS). The protein Spa47 is a T3SS ATPase whose activity is essential for the proper function of the Shigella T3SS needle-like apparatus through which effectors are secreted. A phosphoproteomics study recently found several Shigella T3SS proteins, including Spa47, to be tyrosine phosphorylated, suggesting a means of regulating Spa47 enzymatic activity, T3SS function, and overall Shigella virulence. The work presented here employs phosphomimetic mutations in Spa47 to probe the effects of phosphorylation at these targeted tyrosines through in vitro radiometric ATPase assays and circular dichroism as well as in vivo characterization of T3SS secretion activity, erythrocyte hemolysis, and cellular invasion. Results presented here demonstrate a direct correlation between Spa47 tyrosine phosphorylation state, Spa47 ATPase activity, T3SS function, and Shigella virulence. Together, these findings provide a strong foundation that leads the way to uncovering the specific pathway(s) that Shigella employ to mitigate wasteful ATP hydrolysis and effector protein secretion when not required as well as T3SS activation in preparation for host infection and immune evasion.

High frequency of antimicrobial resistance and virulence gene in Shigella species isolated from pediatric patients in an Iranian Referral Hospital.

Background:Shigella is a main cause of gastroenteritis and it is responsible for 5 to 10 % of diarrhea through the world. The aims of this study were to assess the antibiotic susceptibility pattern and the presence of 3 common virulence genes (sigA, virF, invE) of Shigella strains isolated from patients with gastroenteritis in Children’s Medical Center Hospital, Tehran, Iran.Methods:Over a period of 15 months, all Shigella species collected from the patients with gastroenteritis were entered to the study. Susceptibility testing of all isolates towards different antibiotics was performed using the disk diffusion method and the prevalence of virulence genes was detected by polymerase chain reaction (PCR) technology.Results:Among a total of 183 Shigella strains, 128 Shigella sonnei (70%) and 55 S. flexneri (30%) were isolated. The resistance rate to the antibiotics in S. sonnei strains was higher than S. flexneri. The most sensitive antibiotics for S. flexneri strains were gentamicin (98%), amikacin (85%) and ciprofloxacin (82%), while high resistance rate to trimethoprim-sulfamethoxazole (96%), ampicillin (96%), nalidixic acid (64%) and cefotaxime (60%) was observed. The frequency of invE, virF and sigA gene in S. flexneri strains was 89 %, 93 % and 56 %, respectively; whereas they found in 93 %, 96 %, and 100 % of S. sonnei strains, respectively. SigA gene was identified significantly higher in the S. sonnei strains (100%). There was no significant difference between the presence of virF and invE genes among Shigella strains.Conclusion:The high presence of sigA gene in S. sonnei strains plays an important role in its pathogenesis, and the high frequency of invE and virF genes showed that this classical pathway regulating the expression of Shigella virulence factor genes could play a key role in the pathogenesis of this bacterium. (www.actabiomedica.it)