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 invasion plasmid antigen D (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.

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.

PemB, a type III secretion effector in Pseudomonas aeruginosa, affects Caenorhabditis elegans life span.

Pseudomonas aeruginosa is one of the leading nosocomial opportunistic pathogens causing acute and chronic infections. Among its main virulent factors is the Type III secretion system (T3SS) which enhances disease severity by delivering effectors to the host in a highly regulated manner. Despite its importance for virulence, only six T3SS-dependent effectors have been discovered so far. Previously, we identified two new potential effectors using a machine-learning algorithm approach. Here we demonstrate that one of these effectors, PemB, is indeed virulent. Using a live Caenorhabditis elegans infection model, we demonstrate this effector damages the integrity of the intestine barrier leading to the death of the host. Implementing a high-throughput assay using Saccharomyces cerevisiae, we identified several candidate proteins that interact with PemB. One of them, EFT1, has an ortholog in C. elegans (eef-2) and is also an essential gene and a well-known target utilized by different pathogens to induce toxicity to the worm. Accordingly, we found that by silencing the eef-2 gene in C. elegans, PemB could no longer induce its toxic effect. The current study further uncovers the complex machinery assisting P. aeruginosa virulence and may provide novel insight how to manage infection associated with this hard-to-treat pathogen.

Design and Synthesis of Mandelic Acid Derivatives for Suppression of Virulence via T3SS against Citrus Canker.

Citrus canker, a highly contagious bacterial disease caused by Xanthomonas citri subsp. citri (Xcc), poses a substantial threat to citrus crops, leading to serious reductions in fruit yield and economic losses. Most commonly used bactericides against Xcc lead to the rapid development of resistant subpopulations. Therefore, it is imperative to create novel drugs, such as type III secretion system (T3SS) inhibitors, that specifically target bacterial virulence factors rather than bacterial viability. In our study, we designed and synthesized a series of mandelic acid derivatives including 2-mercapto-1,3,4-thiazole. Seven substances were found to reduce the level of transcription of hpa1 without affecting bacterial viability. In vivo bioassays indicated that compound F9 significantly inhibited hypersensitive response and pathogenicity. RT-qPCR assays showed that compound F9 visibly suppressed the expression of Xcc T3SS-related genes as well as citrus canker susceptibility gene CsLOB1. Furthermore, the combination with compound F9 and quorum-quenching bacteria HN-8 can also obviously alleviate canker symptoms.

New bioactive secondary metabolites from the soil-derived Streptomyces sp. S045 and their anti-bacterial and anti-type III secretion system activities.

A total of seven compounds were isolated from the ISP3 agar cultures of a soil-derived Streptomyces sp. S045 strain. Their structures were determined based on 1D, 2D NMR spectroscopic data, HR ESI mass spectroscopy, X-ray diffraction analysis and comparison with the reported data. The new compounds were identified to be (S)-4-(1-hydroxyethyl)quinoline-2-carboxamide (1) and methyl 4-(hydroxymethyl)-2-(4-methylpentyl)-4,5-dihydrofuran-3-carboxylate (3), respectively. Their anti-bacterial and anti-type III secretion system (T3SS) activities were evaluated.

A novel strategy to control Pseudomonas syringae through inhibition of type III secretion system.

Pseudomonas syringae (P. syringae) is a highly prevalent Gram-negative pathogen with over 60 pathogenic variants that cause yield losses of up to 80% in various crops. Traditional control methods mainly involve the application of antibiotics to inactivate pathogenic bacteria, but large-scale application of antibiotics has led to the development of bacterial resistance. Gram-negative pathogens including P. syringae commonly use the type III secretion system (T3SS) as a transport channel to deliver effector proteins into host cells, disrupting host defences and facilitating virulence, providing a novel target for antibacterial drug development. In this study, we constructed a high-throughput screening reporter system based on our previous work to screen for imidazole, oxazole and thiazole compounds. The screening indicated that the three compounds (II-14, II-15 and II-24) significantly inhibited hrpW and hrpL gene promoter activity without influencing the growth of P. syringae, and the inhibitory activity was better than that of the positive control sulforaphane (4-methylsulfinylbutyl isothiocyanate, SFN) at 50 µM. Three compounds suppressed the transcript levels of representative T3SS genes to different degrees, suggesting that the compounds may suppress the expression of T3SS by modulating the HrpR/S-HrpL regulatory pathway. Inoculation experiments indicated that all three compounds suppressed the pathogenicity of Pseudomonas syringae pv. tomato DC3000 in tomato and Pseudomonas syringae pv. phaseolicola 1448A in bean to varying degrees. One representative compound, II-15, significantly inhibited the secretion of the Pst DC3000 AvrPto effector protein. These findings provide a theoretical basis for the development of novel P. syringae T3SS inhibitors for application in disease prevention and control.

Novel mandelic acid derivatives suppress virulence of Ralstonia solanacearum via type III secretion system.

BACKGROUND: Bacterial wilt induced by Ralstonia solanacearum is regarded as one of the most devastating diseases. However, excessive and repeated use of the same bactericides has resulted in development of bacterial resistance. Targeting bacterial virulence factors, such as type III secretion system (T3SS), without inhibiting bacterial growth is a possible assay to discover new antimicrobial agents. RESULTS: In this work, identifying new T3SS inhibitors, a series of mandelic acid derivatives with 2-mercapto-1,3,4-thiazole moiety was synthesized. One of them, F-24, inhibited the transcription of hrpY gene significantly. The presence of this compound obviously attenuated hypersensitive response (HR) without inhibiting bacterial growth of R. solanacearum. The transcription levels of those typical T3SS genes were reduced to various degrees. The test of the ability of F-24 in protecting plants demonstrated that F-24 protected tomato plants against bacterial wilt without restricting the multiplication of R. solanacearum. The mechanism of this T3SS inhibition is through the PhcR-PhcA-PrhG-HrpB pathway. CONCULSION: The screened F-24 could inhibit R. solanacearum T3SS and showed better inhibitory activity than previously reported inhibitors without affecting the growth of the strain, and F-24 is a compound with good potential in the control of R. solanacearum. © 2023 Society of Chemical Industry.

Pathophysiology of Enteropathogenic Escherichia coli-induced Diarrhea.

Enteropathogenic Escherichia coli (EPEC) are important diarrheal pathogens of infants and young children. Since the availability of molecular diagnosis methods, we now have new insights into the incidence and prevalence of these infections. Recent epidemiological studies indicate that atypical EPEC (aEPEC) are seen more frequently than typical EPEC (tEPEC) worldwide, including in both endemic diarrhea and diarrhea outbreaks. Therefore, it is important to further characterize the pathogenicity of these emerging strains. The virulence mechanisms and pathophysiology of the attaching and effacing lesion (A/E) and the type-three-secretion-system (T3SS) are complex but well-studied. A/E strains use their pool of locus of enterocyte effacement (LEE)-encoded and non-LEE-encoded effector proteins to subvert and modulate cellular and barrier properties of the host. However, the exact mechanisms of diarrhea in EPEC infection are not completely understood. From the clinical perspective, there is a need for fast, easy, and inexpensive diagnostic methods to define optimal treatment and prevention for children in endemic areas. In this article, we present a review of the classification of EPEC, epidemiology, pathogenesis of the disease caused by these bacteria, determinants of virulence, alterations in signaling, determinants of colonization vs. those of disease, and the limited information we have on the pathophysiology of EPEC-induced diarrhea. This article combines peer-reviewed evidence from our own studies and the results of an extensive literature search in the databases PubMed, EMBASE, and Scopus.

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.

Novel Ethyl-3-Aryl-2-Nitroacrylate Derivatives as Potential T3SS Inhibitors against Xanthomonas oryzae pv. oryzae on Rice.

Bacterial leaf blight (BLB) caused by Xanthomonas oryzae pv. oryzae (Xoo) is a highly destructive bacterial disease. Traditional prevention methods have utilized antibiotics to target bacterial growth, which has accelerated the emergence of resistant strains. New prevention techniques are developing agents such as type III secretion system (T3SS) inhibitors that target bacterial virulence factors without affecting bacterial growth. To explore novel T3SS inhibitors, a series of ethyl-3-aryl-2-nitroacrylate derivatives were designed and synthesized. Preliminary screening of T3SS inhibitors was based on the inhibition of the hpa1 gene promoter and showed no effect on bacterial growth. Compounds B9 and B10, obtained in the primary screening, significantly inhibited the hypersensitive response (HR) in tobacco and the expression of T3SS genes in the hrp cluster including key regulatory genes. In vivo bioassays showed that T3SS inhibitors obviously inhibited BLB and appeared to be more effective when combined with quorum quenching bacteria F20.

Novel pyrimidin-4-one derivatives as potential T3SS inhibitors against Xanthomonas campestris pv. campestris.

BACKGROUND: Cruciferous black rot is caused by Xanthomonas campestris pv. campestris (Xcc) infection and is a widespread disease worldwide. Excessive and repeated use of bactericide is an important cause of the development of bacterial resistance. It is imperative to take new approaches to screening compounds that target virulence factors rather than kill bacterial pathogens. The type III secretion system (T3SS) invades a variety of cells by transporting virulence effector factors into the cytoplasm and is an attractive antitoxic target. Toward the search of new T3SS inhibitors, an alternative series of novel pyrimidin-4-one derivatives were designed and synthesized and assessed for their effect in blocking the virulence. RESULTS: All of the target compounds were characterized by proton (1 H) nuclear magnetic resonance (NMR), carbon-13 (13 C) NMR, fluorine-19 (19 F) NMR and high-resolution mass spectrometry (HRMS). All compounds were evaluated using high-throughput screening systems against Xcc. The results of the biological activity test revealed that the compound SPF-9 could highly inhibit the activity of xopN gene promoter and the hypersensitivity (HR) of tobacco without affecting bacterial growth. Moreover, messenger RNA (mRNA) level measurements showed that compound SPF-9 inhibited the expression of some representative genes (hrp/hrc genes). Compound SPF-9 weakened the pathogenicity of Xcc to Raphanus sativus L. CONCLUSION: Compound SPF-9 has good potential for further development as a novel T3SS inhibitor against Xcc. © 2023 Society of Chemical Industry.

Construction of a constitutively active type III secretion system for heterologous protein secretion.

Proteins comprise a multibillion-dollar industry in enzymes and therapeutics, but bacterial protein production can be costly and inefficient. Proteins of interest (POIs) must be extracted from lysed cells and inclusion bodies, purified, and resolubilized, which adds significant time and cost to the protein-manufacturing process. The Salmonella pathogenicity island 1 (SPI-1) type III secretion system (T3SS) has been engineered to address these problems by secreting soluble, active proteins directly into the culture media, reducing the number of purification steps. However, the current best practices method of T3SS pathway activation is not ideal for industrial scaleup. Previously, the T3SS was activated by plasmid-based overexpression of the T3SS transcriptional regulator, hilA, which requires the addition of a small molecule inducer (IPTG) to the culture media. IPTG adds significant cost to production and plasmid-based expression is subject to instability in large-scale fermentation. Here, we modulate the upstream transcriptional regulator, hilD, to activate the T3SS via three distinct methods. In doing so, we develop a toolbox of T3SS activation methods and construct constitutively active T3SS strains capable of secreting a range of heterologous proteins at titers comparable to plasmid-based hilA overexpression. We also explore how each activation method in our toolbox impacts the SPI-1 regulatory cascade and discover an epistatic relationship between T3SS regulators, hilE and the hilD 3' untranslated region (hilD 3'UTR). Together, these findings further our goal of making an industrially competitive protein production strain that reduces the challenges associated with plasmid induction and maintenance. KEY POINTS: • Characterized 3 new type III secretion system (T3SS) activation methods for heterologous protein secretion, including 2 constitutive activation methods. • Eliminated the need for a second plasmid and a small molecule inducer to activate the system, making it more suitable for industrial production. • Discovered new regulatory insights into the SPI-1 T3SS, including an epistatic relationship between regulators hilE and the hilD 3' untranslated region.

Catheter-associated urinary tract infection by Pseudomonas aeruginosa progresses through acute and chronic phases of infection.

Healthcare-associated infections are major causes of complications that lead to extended hospital stays and significant medical costs. The use of medical devices, including catheters, increases the risk of bacterial colonization and infection through the presence of a foreign surface. Two outcomes are observed for catheterized patients: catheter-associated asymptomatic bacteriuria and catheter-associated urinary tract infection (CAUTI). However, the relationship between these two events remains unclear. To understand this relationship, we studied a murine model of Pseudomonas aeruginosa CAUTI. In this model, we also observe two outcomes in infected animals: acute symptoms that is associated with CAUTI and chronic colonization that is associated with asymptomatic bacteriuria. The timing of the acute outcome takes place in the first week of infection, whereas chronic colonization occurs in the second week of infection. We further showed that mutants lacking genes encoding type III secretion system (T3SS), T3SS effector proteins, T3SS injection pore, or T3SS transcriptional activation all fail to cause acute symptoms of CAUTI. Nonetheless, all mutants defective for T3SS colonized the catheter and bladders at levels similar to the parental strain. In contrast, through induction of the T3SS master regulator ExsA, all infected animals showed acute phenotypes with bacteremia. Our results demonstrated that the acute symptoms, which are analogous to CAUTI, and chronic colonization, which is analogous to asymptomatic bacteriuria, are independent events that require distinct bacterial virulence factors. Experimental delineation of asymptomatic bacteriuria and CAUTI informs different strategies for the treatment and intervention of device-associated infections.

Type III effector provides a novel symbiotic pathway in legume-rhizobia symbiosis.

Rhizobia form nodules on the roots of legumes and fix atmospheric nitrogen into ammonia, thus supplying it to host legumes. In return, plants supply photosynthetic products to maintain rhizobial activities. In most cases, rhizobial Nod factors (NFs) and their leguminous receptors (NFRs) are essential for the establishment of symbiosis. However, recent studies have discovered a novel symbiotic pathway in which rhizobia utilize the type III effectors (T3Es) similar to the pathogenic bacteria to induce nodulation. The T3Es of rhizobia are thought to be evolved from the pathogen, but they have a unique structure distinct from the pathogen, suggesting that it might be customized for symbiotic purposes. This review will focus on the recent findings from the study of rhizobial T3Es, discussing their features on a symbiont and pathogen, and the future perspectives on the role of rhizobial T3Es in symbiosis control technology.

Insight Into Distinct Functional Roles of the Flagellar ATPase Complex for Flagellar Assembly in Salmonella.

Most motile bacteria utilize the flagellar type III secretion system (fT3SS) to construct the flagellum, which is a supramolecular motility machine consisting of basal body rings and an axial structure. Each axial protein is translocated via the fT3SS across the cytoplasmic membrane, diffuses down the central channel of the growing flagellar structure and assembles at the distal end. The fT3SS consists of a transmembrane export complex and a cytoplasmic ATPase ring complex with a stoichiometry of 12 FliH, 6 FliI and 1 FliJ. This complex is structurally similar to the cytoplasmic part of the FOF1 ATP synthase. The export complex requires the FliH12-FliI6-FliJ1 ring complex to serve as an active protein transporter. The FliI6 ring has six catalytic sites and hydrolyzes ATP at an interface between FliI subunits. FliJ binds to the center of the FliI6 ring and acts as the central stalk to activate the export complex. The FliH dimer binds to the N-terminal domain of each of the six FliI subunits and anchors the FliI6-FliJ1 ring to the base of the flagellum. In addition, FliI exists as a hetero-trimer with the FliH dimer in the cytoplasm. The rapid association-dissociation cycle of this hetero-trimer with the docking platform of the export complex promotes sequential transfer of export substrates from the cytoplasm to the export gate for high-speed protein transport. In this article, we review our current understanding of multiple roles played by the flagellar cytoplasmic ATPase complex during efficient flagellar assembly.

Insights into the structure-activity relationship of a type III secretion system inhibitor, aurodox.

Aurodox was originally isolated in 1972 as a linear polyketide compound exhibiting antibacterial activity against Gram-positive bacteria. We have since identified aurodox as a specific inhibitor of the bacterial type III secretion system (T3SS) using our original screening system for inhibition of T3SS-mediated hemolysis in enteropathogenic Escherichia coli (EPEC). In this research, we synthesized 15 derivatives of aurodox and evaluated EPEC T3SS inhibitory activity as well as antibacterial activity against EPEC. One of the derivatives was highly selective for T3SS inhibition, equivalent to that of aurodox, but without exhibiting antibacterial activity (69-fold selectivity). This work revealed the structure-activity relationship for the inhibition of T3SS by aurodox and suggests that the target of T3SS is distinct from the target for antibacterial activity.

Calmodulin Binding Activates Chromobacterium CopC Effector to ADP-Riboxanate Host Apoptotic Caspases.

Blocking host cell death is an important virulence strategy employed by many bacterial pathogens. We recently reported that Shigella flexneri inhibits host pyroptosis by delivering a type III secretion system (T3SS) effector OspC3 that catalyzes a novel arginine ADP-riboxanation modification on caspase-4/11. Here, we investigated the OspC3 homologue CopC from Chromobacterium violaceum, an opportunistic but sometimes deadly bacterial pathogen. CopC bears the same arginine ADP-riboxanase activity as OspC3, but with a different substrate specificity. Through proteomic analysis, we first identified host calmodulin (CaM) as a binding partner of CopC. The analyses additionally revealed that CopC preferably modifies apoptotic caspases including caspase-7, -8 and -9. This results in suppression of both extrinsic and intrinsic apoptosis programs in C. violaceum-infected cells. Biochemical reconstitution showed that CopC requires binding to CaM, specifically in the calcium-free state, to achieve efficient ADP-riboxanation of the caspases. We determined crystal structure of the CaM-CopC-CASP7 ternary complex, which illustrates the caspase recognition mechanism and a unique CaM-binding mode in CopC. Structure-directed mutagenesis validated the functional significance of CaM binding for stimulating CopC modification of its caspase substrates. CopC adopts an ADP-ribosyltransferase-like fold with a unique His-Phe-Glu catalytic triad, featuring two acidic residues critical for site-specific arginine ADP-riboxanation. Our study expands and deepens our understanding of the OspC family of ADP-riboxanase effectors. IMPORTANCE Programmed cell death is a suicidal defense mechanism for eukaryotes to combat pathogen infection. In the evolutionary arms race with the host, bacteria are endowed with ingenious tactics to block host cell death to facilitate their replication. Here, we report that the C. violaceum effector CopC ADP-riboxanates caspase-7/8/9, enabled by interacting with the host factor calmodulin, to block host cell apoptosis, illustrating a unique and sophisticated strategy adopted by the pathogen to counteract host defense.

A small molecule, C24H17ClN4O2S, inhibits the function of the type III secretion system in Salmonella Typhimurium.

BACKGROUND: Salmonella enterica serovar Typhimurium (S. Typhimurium) causes gastroenteritis and diarrhea in humans and food-producing animals. The type III secretion system (T3SS) has been known to be a potent virulence mechanism by injecting effector proteins into the cytosol of host cells. S. Typhimurium encodes two T3SSs by Salmonella pathogenicity islands 1 and 2. Previous studies showed that T3SS shared a potent virulence mechanism and molecular structure among several gram-negative bacteria. Therefore, T3SS has been identified as an attractive target in the development of novel therapeutics for the treatment of bacterial infections. Several studies reported that small-molecule compounds are able to inhibit functions of bacterial T3SSs. A small molecule, C24H17ClN4O2S, has been shown the ability to inhibit the activity of Yersinia pestis T3SS ATPase, YscN, resulting to block the secretion of effector proteins. In this study, we studied the effects and mechanism for SPI-1 T3SS inhibition of this compound in S. Typhimurium. RESULTS: We demonstrated that this compound prohibited the secretion of effector proteins from Salmonella via SPI-1 T3SS at 100 µM. As the result, bacterial invasion ability into epithelial cell cultures was reduced. In contrast with previous study, the C24H17ClN4O2S molecule did not inactivate the activity of SPI-1 T3SS ATPase, InvC, in Salmonella. However, we studied the global cellular effects of S. Typhimurium after being treated with this compound using a quantitative proteomic technique. These proteomic results showed that the main SPI-1 transcription regulator, InvF, and two effector proteins, SipA and SipC, were reduced in bacterial cells treated with the compound. CONCLUSIONS: It may explain that action of the small-molecule compound, C24H17ClN4O2S, for blocking the secretion of SPI-1 T3SS in Salmonella is through inhibition of SPI-1 regulator, InvF, expression. Further studies are necessary to identify specific mechanisms for inhibition between this small-compound and InvF SPI-1 regulator protein.

Bifunctional alcohol/aldehyde dehydrogenase AdhE controls phospho-transferase system sugar utilization and virulence gene expression by interacting PtsH in Edwardsiella piscicida.

The bifunctional alcohol/aldehyde dehydrogenase (AdhE), one of the key enzymes in the bacterial ethanol anaerobic fermentation pathway, is critical for appropriate expression of the genes for the utilization of carbon sources. Knowledge about its global roles in modulating gene expression and metabolomics remains limited. Edwardsiella bacteria includes several important zoonotic pathogenic species including Edwardsiella piscicida, a leading fish pathogen that causes severe economic losses in the aquaculture industry. It is well known to utilize few sugars. In this study, we showed that AdhE is involved in various processes including sugar utilization, bacteria growth, intracellular pH homeostasis, type III/VI secretion system (T3/T6SS) production, and survival in fish. Moreover, our unbiased metabolomics approaches revealed that AdhE modulates a large quantity of metabolic pathways, including amino acids, tricarboxylic acid (TCA) intermediates, sugar and fatty acids. Pull-down and Co-immunoprecipitation (IP) analysis revealed that AdhE interacts with the phospho-transferase system component PtsH that supports the transform of its PTS sugars including mannose to mannose-6P, the established metabolic ligand modulating EvrA activity to control T3/T6SS expression. Collectively, AdhE appears to play important roles in bacterial adapting to the internal environment changes by regulating sugar metabolic pathways and bacterial virulence expression. These observations support a model in which AdhE acts a macromolecule hub accommodating proteins to modulate the PTS and other signaling cascades related to pathogenesis and environmental adaptation in bacterial pathogens, which may provide new perspectives for attempts to attenuate bacterial virulence.

Coordinate regulation of carbohydrate metabolism and virulence by PtsH in pathogen Edwardsiella piscicida.

Carbohydrate metabolism of bacterial pathogens conducts crucial roles in regulating pathogenesis but the molecular mechanisms by which metabolisms and virulence are been modulated and coordinated remain to be illuminated. Here, we investigated in this regard Edwardsiella piscicida, a notorious zoonotic pathogen previously named E. tarda that could ferment very few PTS sugars including glucose, fructose, mannose, N-acetylglucosamine, and N-acetylgalactosamine. We systematically characterized the roles of each of the predicted 23 components of phosphotransferase system (PTS) with the respective in-frame deletion mutants and defined medium containing specific PTS sugar. In addition, PtsH was identified as the crucial PTS component potentiating the utilization of all the tested PTS sugars. Intriguingly, we also found that PtsH while not Fpr was involved in T3SS gene expression and was essential for the pathogenesis of E. piscicida. To corroborate this, His15 and Ser46, the two established PtsH residues involved in phosphorylation cascade, showed redundant roles in regulating T3SS yields. Moreover, PtsH was shown to facilitate mannose uptake and transform it into mannose-6-phosphate, an allosteric substrate established to activate EvrA to augment bacterial virulence. Collectively, our observations provide new insights into the roles of PTS reciprocally regulating carbohydrate metabolism and virulence gene expression. KEY POINTS: • PTS components' roles for sugar uptake are systematically determined in Edwardsiella piscicida. • PtsH is involved in saccharides uptake and in the regulation of E. piscicida's T3SS expression. • PtsH phosphorylation at His15 and Ser46 is essential for the T3SS expression and virulence.