Impact of Two Phosphorus Fertilizer Formulations on Wheat Physiology, Rhizosphere, and Rhizoplane Microbiota.

Phosphorus (P) is the second most important macronutrient for crop growth and a limiting factor in food production. Choosing the right P fertilizer formulation is important for crop production systems because P is not mobile in soils, and placing phosphate fertilizers is a major management decision. In addition, root microorganisms play an important role in helping phosphorus fertilization management by regulating soil properties and fertility through different pathways. Our study evaluated the impact of two phosphorous formulations (polyphosphates and orthophosphates) on physiological traits of wheat related to yield (photosynthetic parameters, biomass, and root morphology) and its associated microbiota. A greenhouse experiment was conducted using agricultural soil deficient in P (1.49%). Phenotyping technologies were used at the tillering, stem elongation, heading, flowering, and grain-filling stages. The evaluation of wheat physiological traits revealed highly significant differences between treated and untreated plants but not between phosphorous fertilizers. High-throughput sequencing technologies were applied to analyse the wheat rhizosphere and rhizoplane microbiota at the tillering and the grain-filling growth stages. The alpha- and beta-diversity analyses of bacterial and fungal microbiota revealed differences between fertilized and non-fertilized wheat, rhizosphere, and rhizoplane, and the tillering and grain-filling growth stages. Our study provides new information on the composition of the wheat microbiota in the rhizosphere and rhizoplane during growth stages (Z39 and Z69) under polyphosphate and orthophosphate fertilization. Hence, a deeper understanding of this interaction could provide better insights into managing microbial communities to promote beneficial plant-microbiome interactions for P uptake.

Nod1 and Nod2 direct autophagy by recruiting ATG16L1 to the plasma membrane at the site of bacterial entry.

Autophagy is emerging as a crucial defense mechanism against bacteria, but the host intracellular sensors responsible for inducing autophagy in response to bacterial infection remain unknown. Here we demonstrated that the intracellular sensors Nod1 and Nod2 are critical for the autophagic response to invasive bacteria. By a mechanism independent of the adaptor RIP2 and transcription factor NF-kappaB, Nod1 and Nod2 recruited the autophagy protein ATG16L1 to the plasma membrane at the bacterial entry site. In cells homozygous for the Crohn's disease-associated NOD2 frameshift mutation, mutant Nod2 failed to recruit ATG16L1 to the plasma membrane and wrapping of invading bacteria by autophagosomes was impaired. Our results link bacterial sensing by Nod proteins to the induction of autophagy and provide a functional link between Nod2 and ATG16L1, which are encoded by two of the most important genes associated with Crohn's disease.

Functional insights into the Shigella type III needle tip IpaD in secretion control and cell contact.

Type III secretion apparatus (T3SA) are complex nanomachines that insert a translocation pore into the host cell membrane through which effector proteins are injected into the cytosol. In Shigella, the pore is inserted by a needle tip complex that also controls secretion. IpaD is the key protein that rules the composition of the tip complex before and upon cell contact or Congo red (CR) induction. However, how IpaD is involved in secretion control and translocon insertion remains not fully understood. Here, we report the phenotypic analysis of 20 10-amino acids deletion variants all along the coiled-coil and the central domains of IpaD (residues 131-332). Our results highlight three classes of T3S phenotype; (i) wild-type secretion, (ii) constitutive secretion of all classes of effectors, and (iii) constitutive secretion of translocators and early effectors, but not of late effectors. Our data also suggest that the composition of the tip complex defines both the T3SA inducibility state and late effectors secretion. Finally, we shed light on a new aspect regarding the contact of the needle tip with cell membrane by uncoupling the Shigella abilities to escape macrophage vacuole, and to insert the translocation pore or to invade non-phagocytic cells.

Interplay between predicted inner-rod and gatekeeper in controlling substrate specificity of the type III secretion system.

The type III secretion apparatus (T3SA) is a multi-protein complex central to the virulence of many Gram-negative pathogens. Currently, the mechanisms controlling the hierarchical addressing of needle subunits, translocators and effectors to the T3SA are still poorly understood. In Shigella, MxiC is known to sequester effectors within the cytoplasm prior to receiving the activation signal from the needle. However, molecules involved in linking the needle and MxiC are unknown. Here, we demonstrate a molecular interaction between MxiC and the predicted inner-rod component MxiI suggesting that this complex plugs the T3SA entry gate. Our results suggest that MxiI-MxiC complex dissociation facilitates the switch in secretion from translocators to effectors. We identified MxiC(F)(206)(S) variant, unable to interact with MxiI, which exhibits a constitutive secretion phenotype although it remains responsive to induction. Moreover, we identified the mxiI(Q67A) mutant that only secretes translocators, a phenotype that was suppressed by coexpression of the MxiC(F)(206)(S) variant. We demonstrated the interaction between MxiI and MxiC homologues in Yersinia and Salmonella. Lastly, we identified an interaction between MxiC and chaperone IpgC which contributes to understanding how translocators secretion is regulated. In summary, this study suggests the existence of a widely conserved T3S mechanism that regulates effectors secretion.

Spa13 of Shigella flexneri has a dual role: chaperone escort and export gate-activator switch of the type III secretion system.

The type III secretion apparatus (T3SA) is used by numerous Gram-negative pathogens to inject virulence factors into eukaryotic cells. The Shigella flexneri T3SA spans the bacterial envelope and its assembly requires the products of ~20 mxi and spa genes. Despite progress made in understanding how the T3SA is assembled, the role of several predicted soluble components, such as Spa13, remains elusive. Here, we show that the secretion defect of the spa13 mutant is associated with lack of T3SA assembly which is partly due to the instability of the needle component MxiH. In contrast to its Yersinia counterpart, Spa13 is not a secreted protein. We identified a network of interactions between Spa13 and the ATPase Spa47, the C-ring protein Spa33, and the inner-membrane protein Spa40. Moreover, we revealed a Spa13 interaction with the inner-membrane MxiA and showed that overexpression of the large cytoplasmic domain of MxiA in the WT background shuts off secretion. Lastly, we demonstrated that Spa13 interacts with the cleaved form of Spa40 and with the translocator chaperone IpgC, suggesting that Spa13 intervenes during the secretion hierarchy switch process. Collectively, our results support a dual role of Spa13 as a chaperone escort and as an export gate-activator switch.

Development of a visual Adhesion/Invasion Inhibition Assay to assess the functionality of Shigella-specific antibodies.

Introduction: Shigella is the etiologic agent of a bacillary dysentery known as shigellosis, which causes millions of infections and thousands of deaths worldwide each year due to Shigella's unique lifestyle within intestinal epithelial cells. Cell adhesion/invasion assays have been extensively used not only to identify targets mediating host-pathogen interaction, but also to evaluate the ability of Shigella-specific antibodies to reduce virulence. However, these assays are time-consuming and labor-intensive and fail to assess differences at the single-cell level. Objectives and methods: Here, we developed a simple, fast and high-content method named visual Adhesion/Invasion Inhibition Assay (vAIA) to measure the ability of anti-Shigellaantibodies to inhibit bacterial adhesion to and invasion of epithelial cells by using the confocal microscope Opera Phenix. Results: We showed that vAIA performed well with a pooled human serum from subjects challenged with S. sonnei and that a specific anti-IpaD monoclonal antibody effectively reduced bacterial virulence in a dose-dependent manner. Discussion: vAIA can therefore inform on the functionality of polyclonal and monoclonal responses thereby supporting the discovery of pathogenicity mechanisms and the development of candidate vaccines and immunotherapies. Lastly, this assay is very versatile and may be easily applied to other Shigella species or serotypes and to different pathogens.

Nanobody-Based Sandwich Immunoassay for Pathogenic Escherichia coli F17 Strain Detection.

Rapid and specific detection of pathogenic bacteria in fecal samples is of critical importance for the diagnosis of neonatal diarrhea in veterinary clinics. Nanobodies are a promising tool for the treatment and diagnosis of infectious diseases due to their unique recognition properties. In this study, we report the design of a nanobody-based magnetofluorescent immunoassay for the sensitive detection of pathogenic Escherichia coli F17-positive strains (E. coli F17). For this, a camel was immunized with purified F17A protein from F17 fimbriae and a nanobody library was constructed by phage display. Two specific anti-F17A nanobodies (Nbs) were selected to design the bioassay. The first one (Nb1) was conjugated to magnetic beads (MBs) to form a complex capable of efficiently capturing the target bacteria. A second horseradish peroxidase (HRP)-conjugated nanobody (Nb4) was used for detection by oxidizing o-phenylenediamine (OPD) to fluorescent 2,3-diaminophenazine (DAP). Our results show that the immunoassay recognizes E. coli F17 with high specificity and sensitivity, with a detection limit of 1.8 CFU/mL in only 90 min. Furthermore, we showed that the immunoassay can be applied to fecal samples without pretreatment and remains stable for at least one month when stored at 4 °C.

Bacillus velezensis QA2 Potentially Induced Salt Stress Tolerance and Enhanced Phosphate Uptake in Quinoa Plants.

Plant Growth-Promoting Rhizobacteria (PGPR) have attracted much attention in agriculture biotechnology as biological inputs to sustain crop production. The present study describes a halotolerant phosphate solubilizing bacterium associated with quinoa plant roots. Based on a metabolic screening, one bacterial isolate, named QA2, was selected and screened for PGPR traits. This isolate solubilized both inorganic phosphate and zinc, produced indole-3-acetic acid, ammonia, hydrogen cyanide, cellulase, and (to be deleted) protease, and induced biofilm formation. We demonstrated that QA2 exhibited both antimicrobial and ion metabolism activities and tolerated high salt concentration at up to 11% NaCl. Genotyping analyses, using 16S rRNA and chaperonin cpn60 genes, revealed that QA2 belongs to the species of Bacillus velezensis. Using the quinoa model cultivated under a saline condition, we demonstrated that QA2 promoted plant growth and mitigated the saline irrigation effects. Analysis of harvested plants revealed that QA2 induced a significant increase of both leaf chlorophyll index by 120.86% (p < 0.05) and P uptake by 41.17% (p < 0.05), while the content of Na+ was drastically decreased. Lastly, a bibliometric data analysis highlighted the panoramic view of studies carried out so far on B. velezensis strains. Our investigation presents a holistic view of the potential application of B. velezensis as a biological inoculant to promote plant growth, control pathogen attacks, and mitigate the salinity effect of quinoa plants. Further investigations are still needed to demonstrate these effects in field conditions.

Phosphate solubilizing rhizobacteria isolated from jujube ziziphus lotus plant stimulate wheat germination rate and seedlings growth.

Jujube plant (Ziziphus lotus (L.) Desf.) can survive in arid climates and tolerates both biotic and abiotic stresses. Here, we isolated, for the first time in Morocco, nine phosphate solubilizing bacteria strains from jujube rhizosphere, designated J10 to J13, J15, & J153 to J156. Genotypic identification based on 16S rDNA sequencing, revealed six strains that belong to Pseudomonas (J10, J12, J13, J15, J153 and J154), two to Bacillus (J11 and J156), and one to Paenibacillus J155. Siderophores were produced by all strains. Proteases activity was missing in Pseudomonas sp. J153 & J154, whereas cellulase was restricted only to Pseudomonas sp. J10, Paenibacillus xylanexedens J155 and Bacillus cereus J156. Indole-3- acetic acid and ammonia were also produced by all strains, with a maxima of 204.28 µg mL-1 in Bacillus megaterium J11 and 0.33 µmol mL-1 in Pseudomonas sp. J153, respectively. Pseudomonas sp. J10 and B. cereus J156 grew on plates containing 1,500 µg mL-1 of nickel nitrate, while Pseudomonas sp. J153 withstood 1,500 µg mL-1 of either copper sulfate or cadmium sulfate. Phenotypic analysis of the potential of the isolates to promote early plant growth showed that wheat seeds inoculated with either P. moraviensis J12 or B. cereus J156 remarkably increased germination rate and seedlings growth. Lastly, antibiotic resistance profiling revealed that except for Pseudomonas sp. J11 and B. cereus J156, remaining strains displayed resistance at least to one of tested antibiotics. Collectively, Pseudomonas sp. J10, P. moraviensis J12, Pseudomonas sp. J153 and B. cereus J156, represent potential biofertilizers suitable for soils that are poor in P, and/or heavy metals contaminated.

MxiC is secreted by and controls the substrate specificity of the Shigella flexneri type III secretion apparatus.

Many gram-negative pathogenic bacteria use a type III secretion (T3S) system to interact with cells of their hosts. Mechanisms controlling the hierarchical addressing of needle subunits, translocators and effectors to the T3S apparatus (T3SA) are still poorly understood. We investigated the function of MxiC, the member of the YopN/InvE/SepL family in the Shigella flexneri T3S system. Inactivation of mxiC led specifically to a deregulated secretion of effectors (including IpaA, IpgD, IcsB, IpgB2, OspD1 and IpaHs), but not of translocators (IpaB and IpaC) and proteins controlling the T3SA structure or activity (Spa32 and IpaD). Expression of effector-encoding genes controlled by the activity of the T3SA and the transcription activator MxiE was increased in the mxiC mutant, as a consequence of the increased secretion of the MxiE anti-activator OspD1. MxiC is a T3SA substrate and its ability to be secreted is required for its function. By using co-purification assays, we found that MxiC can associate with the Spa47 ATPase, which suggests that MxiC might prevent secretion of effectors by blocking the T3SA from the inside. Although with a 10-fold reduced efficiency compared with the wild-type strain, the mxiC mutant was still able to enter epithelial cells.

The 33 carboxyl-terminal residues of Spa40 orchestrate the multi-step assembly process of the type III secretion needle complex in Shigella flexneri.

The type III secretion apparatus (T3SA) is a central virulence factor of many Gram-negative bacteria. Its overall morphology consists of a cytoplasmic region, inner- and outer-membrane sections and an extracellular needle. In Shigella, the length of the needle is regulated by Spa32. To understand better the role of Spa32 we searched for its interacting partners using a two-hybrid screen in yeast. We found that Spa32 interacts with the 33 C-terminal residues (CC*) of Spa40, a member of the conserved FlhB/YscU family. Using a GST pull-down assay we confirmed this interaction and identified additional interactions between Spa40 and the type III secretion components Spa33, Spa47, MxiK, MxiN and MxiA. Inactivation of spa40 abolished protein secretion and led to needleless structures. Genetic and functional analyses were used to investigate the roles of residues L310 and V320, located within the CC* domain of Spa40, in the assembly of the T3SA. Spa40 cleavage, at the conserved NPTH motif, is required for assembly of the T3SA and for its interaction with Spa32, Spa33 and Spa47. In contrast, unprocessed forms of Spa40 interacted only with MxiA, MxiK and MxiN. Our data suggest that the conformation of the cytoplasmic domain of Spa40 defines the multi-step assembly process of the T3SA.

Inactivation of the sfgtr4 Gene of Shigella flexneri Induces Biofilm Formation and Affects Bacterial Pathogenicity.

Biofilm formation is a significant cause for the environmental persistence of foodborne pathogens. This phenomenon remains misunderstood in Shigella flexneri whose pathogenicity is mainly associated with the virulence plasmid pWR100. Sequence analysis of the latter predicts a putative lipopolysaccharides (LPS) glycosyltransferase (Gtr) encoded by Sfgtr4, which is the second gene of the SfpgdA-orf186-virK-msbB2 locus. We demonstrated here that purified SfGtr4 exhibited a Gtr activity in vitro by transferring glucose to lipid A. To establish the role of SfGtr4 in virulence, we generated a Sfgtr4 mutant and assessed its phenotype in vitro. Sfgtr4 mutant significantly reduced HeLa cells invasion without impairing type III effectors secretion, increased susceptibility to lysozyme degradation, and enhanced bacterial killing by polymorphonuclear neutrophils (PMNs). SfGtr4 is related to proteins required in biofilm formation. We established conditions whereby wild-type Shigella formed biofilm and revealed that its appearance was accelerated by the Sfgtr4 mutant. Additional phenotypical analysis revealed that single SfpdgA and double SfpgdA-Sfgtr4 mutants behaved similarly to Sfgtr4 mutant. Furthermore, a molecular interaction between SfGtr4 and SfPgdA was identified. In summary, the dual contribution of SfGtr4 and SfPgdA to the pathogenicity and the regulation biofilm formation by S. flexneri was demonstrated here.

Plant Growth Enhancement using Rhizospheric Halotolerant Phosphate Solubilizing Bacterium Bacillus licheniformis QA1 and Enterobacter asburiae QF11 Isolated from Chenopodium quinoa Willd.

Plant growth-promoting rhizobacteria represent a promising solution to enhancing agricultural productivity. Here, we screened phosphate solubilizing bacteria from the rhizospheric soil of Chenopodium quinoa Willd and assessed their plant-growth promoting rhizobacteria (PGPR) properties including production of indole-3-acetic acid (IAA), siderophores, hydrogen cyanide (HCN), ammonia and extracellular enzymes. We also investigated their tolerance to salt stress and their capacity to form biofilms. Two isolated strains, named QA1 and QF11, solubilized phosphate up to 346 mg/L, produced IAA up to 795.31 µg/mL, and tolerated up to 2 M NaCl in vitro. 16S rRNA and Cpn60 gene sequencing revealed that QA1 and QF11 belong to the genus Bacillus licheniformis and Enterobacter asburiae, respectively. In vivo, early plant growth potential showed that quinoa seeds inoculated either with QA1 or QF11 displayed higher germination rates and increased seedling growth. Under saline irrigation conditions, QA1 enhanced plant development/growth. Inoculation with QA1 increased leaf chlorophyll content index, enhanced P and K+ uptake and decreased plant Na+ uptake. Likewise, plants inoculated with QF11 strain accumulated more K+ and had reduced Na+ content. Collectively, our findings support the use of QA1 and QF11 as potential biofertilizers.

Spa32 interaction with the inner-membrane Spa40 component of the type III secretion system of Shigella flexneri is required for the control of the needle length by a molecular tape measure mechanism.

The effectors of enterocyte invasion by Shigella are dependent on a type III secretion system that contains a needle whose length average does not exceed 50 nm. Previously, we reported that Spa32 is required for needle length control as well as to switch substrate specificity from MxiH to Ipa proteins secretion. To identify functional domains of Spa32, 11 truncated variants were constructed and analysed for their capacity (i) to control the needle's length; (ii) to secrete the Ipa proteins; and (iii) to invade HeLa cells. Deletion at either the N-terminus or C-terminus affect Spa32 function in all cases, but Spa32 variants lacking internal residues 37-94 or 130-159 retained full Spa32 function. Similarly, a Spa32 variant obtained by inserting of the YscP's ruler domain retained Spa32 function although it programmed slightly elongated needles. Using the GST pull-down assay, we show that residues 206-246 are required for Spa32 binding to the C-terminus of Spa40, an inner membrane protein required for Ipa proteins secretion. Our data clearly demonstrate that shortening Spa32 affects the length of the needle in a comparable manner to the spa32 mutant, indicating that the control of needle length does not require a molecular ruler mechanism.

IpaD is localized at the tip of the Shigella flexneri type III secretion apparatus.

Type III secretion (T3S) systems are used by numerous Gram-negative pathogenic bacteria to inject virulence proteins into animal and plant host cells. The core of the T3S apparatus, known as the needle complex, is composed of a basal body transversing both bacterial membranes and a needle protruding above the bacterial surface. In Shigella flexneri, IpaD is required to inhibit the activity of the T3S apparatus prior to contact of bacteria with host and has been proposed to assist translocation of bacterial proteins into host cells. We investigated the localization of IpaD by electron microscopy analysis of cross-linked bacteria and mildly purified needle complexes. This analysis revealed the presence of a distinct density at the needle tip. A combination of single particle analysis, immuno-labeling and biochemical analysis, demonstrated that IpaD forms part of the structure at the needle tip. Anti-IpaD antibodies were shown to block entry of bacteria into epithelial cells.

Expression of galectin-3 in the tumor immune response in colon cancer.

The role of tumor-associated macrophages (TAMs) is controversial. Although most studies on different cancer types associate them with a poorer prognosis, interestingly in colon cancer, most articles indicate that TAMs prevent tumor development; patients with high TAMs have better prognosis and survival rate. M1-polarized macrophages produce high level of tumor necrosis factor-alpha, interleukin-1 beta or reactive oxygen species, which can effectively kill susceptible tumor cells. In contrast, M2-polarized macrophages can secrete different factors that promote tumor cell growth and survival or favor angiogenesis and tissue invasion. Considering the beneficial role of TAMs in colon cancer, we speculated that they may not display the M2 polarization commonly observed in tumor microenvironment, but rather develop M1 properties. Therefore, we used an in vitro model to analyze the effects of supernatants from M1-polarized macrophages on DLD-1 colon cancer cells. Our data indicate that the conditioned medium from LPS-activated macrophages (CM-LAM) contains a high level of granulocyte-macrophage colony-stimulating factor, interleukins-1 beta, -6, -8 and tumor necrosis factor-alpha, and that it exerts a marked growth inhibitory activity on DLD-1 cells. Prolonged exposure to CM-LAM results in cell death by apoptosis. Such exposure to CM-LAM leads to the modulation of gal-3 expression: we observed a marked downregulation of gal-3 mRNA and protein expression following CM-LAM treatment. We also describe that the knockdown of gal-3 sensitizes DLD-1 cells to CM-LAM. These data suggest an involvement of gal-3 in the response of colon cancer cells to proinflammatory stimuli, such as the conditioned medium from activated macrophages.

IpgB1 and IpgB2, two homologous effectors secreted via the Mxi-Spa type III secretion apparatus, cooperate to mediate polarized cell invasion and inflammatory potential of Shigella flexenri.

Type III secretion systems (T3SS) are present in many pathogenic gram-negative bacteria and mediate the translocation of bacterial effector proteins into host cells. Here, we report the phenotypic characterization of S. flexneri ipgB1 and ipgB2 mutants, in which the genes encoding the IpgB1 and IpgB2 effectors have been inactivated, either independently or simultaneously. Like IpgB1, we found that IpgB2 is secreted by the T3SS and its secretion requires the Spa15 chaperone. Upon infection of semi-confluent HeLa cells, the ipgB2 mutant exhibited the same invasive capacity as the wild-type strain and the ipgB1 mutant was 50% less invasive. Upon infection of polarised Caco2-cells, the ipgB2 mutant did not show a significant defect in invasion and the ipgB1 mutant was slightly more invasive than the wild-type strain. Entry of the ipgB1 ipgB2 mutant in polarized cells was reduced by 70% compared to the wild-type strain. Upon infection of the cornea in Guinea pigs, the ipgB2 mutant exhibited a wild-type phenotype, the ipgB1 mutant was hypervirulent and elicited a more pronounced proinflammatory response, while the ipgB1 ipgB2 mutant was highly attenuated. The attenuated phenotype of the ipgB1 ipgB2 mutant was confirmed using a murine pulmonary model of infection and histopathology and immunochemistry studies.

Structural organization of the needle complex of the type III secretion apparatus of Shigella flexneri.

The secretion apparatus known as the needle complex (NC) from the bacterium Shigella flexneri was studied by single particle electron microscopy. The isolated intact NC appears in projection to be composed of a basal body consisting of seven rings and a protruding needle appendage. A comparison of averaged projections of the intact NC and its fragments revealed the organization of the NC into several major subcomplexes. One of these lacks an inner membrane ring of the basal body but still presents the needle appendage attached to four upper rings. The position of the needle appendage within these rings is variable, suggesting that the dissociated component is necessary for stabilizing the needle appendage. Averaged images of the subcomplex lacking the inner membrane basal rings show a thicker extension at the base of the needle appendage, called the socket. This socket was also found to be present in images of the basal body fragment isolated from mutants lacking the mxiH and mxiI genes. This suggests that the socket is not composed of MxiH and MxiI subunits, which form the needle appendage. A symmetry analysis of the basal body top view projections indicated that a peripheral protein component of the inner membrane ring is present in a ring with 24 copies, in contrast to the Salmonella typhimurium NC. A model is presented in which the NC is only associated to the outer- and inner-membranes with its first and seventh ring, respectively.

Shigella stands up to the challenge of adhesion.

The invasion process of S. flexneri is well characterized, but mechanisms underlying this bacterium's adhesion to host cells have remained obscure. In this issue of Cell Host & Microbe, Brotcke Zumsteg et al. (2014) report a surprising role for the Shigella virulence factor IcsA (VirG) as an adhesin.

Single amino acid substitutions on the needle tip protein IpaD increased Shigella virulence.

Infection of colonic epithelial cells by Shigella is associated with the type III secretion system, which serves as a molecular syringe to inject effectors into host cells. This system includes an extracellular needle used as a conduit for secreted proteins. Two of these proteins, IpaB and IpaD, dock at the needle tip to control secretion and are also involved in the insertion of a translocation pore into host cell membrane allowing effector delivery. To better understand the function of IpaD, we substituted thirteen residues conserved among homologous proteins in other bacterial species. Generated variants were tested for their ability to surface expose IpaB and IpaD, to control secretion, to insert the translocation pore, and to invade host cells. In addition to a first group of seven ipaD variants that behaved similarly to the wild-type strain, we identified a second group with mutations V314D and I319D that deregulated secretion of all effectors, but remained fully invasive. Moreover, we identified a third group with mutations Y153A, T161D, Q165L and Y276A, that exhibited increased levels of translocators secretion, pore formation, and cell entry. Altogether, our results offer a better understanding of the role of IpaD in the control of Shigella virulence.