Total synthesis of the antibacterial polyketide natural product thailandamide lactone.

Stereoselective total synthesis of the structurally intriguing polyketide natural product thailandamide lactone was accomplished, and done so using a convergent approach for the first time to the best of our knowledge. The key features of this synthesis included use of a Crimmins acetate aldol reaction, Evans methylation, Urpi acetal aldol reaction, Sharpless asymmetric epoxidation and subsequent γ-lactonization for the installation of six asymmetric centers and the use of the Negishi reaction, Julia-Kocienski olefination, cross metathesis, HWE olefination and intermolecular Heck coupling for construction of a variety of unsaturated linkages. Pd(i)-based Heck coupling was introduced, for the first time to the best of our knowledge, quite efficiently to couple the major eastern and sensitive western segments of the molecule. The antibacterial activity of thailandamide lactone was also evaluated.

Discovery of a Novel Inner Membrane-Associated Bacterial Structure Related to the Flagellar Type III Secretion System.

The bacterial flagellar type III secretion system (fT3SS) is a suite of membrane-embedded and cytoplasmic proteins responsible for building the flagellar motility machinery. Homologous nonflagellar (NF-T3SS) proteins form the injectisome machinery that bacteria use to deliver effector proteins into eukaryotic cells, and other family members were recently reported to be involved in the formation of membrane nanotubes. Here, we describe a novel, evolutionarily widespread, hat-shaped structure embedded in the inner membranes of bacteria, of yet-unidentified function, that is present in species containing fT3SS. Mutant analysis suggests a relationship between this novel structure and the fT3SS, but not the NF-T3SS. While the function of this novel structure remains unknown, we hypothesize that either some of the fT3SS proteins assemble within the hat-like structure, perhaps including the fT3SS core complex, or that fT3SS components regulate other proteins that form part of this novel structure. IMPORTANCE The type III secretion system (T3SS) is a fascinating suite of proteins involved in building diverse macromolecular systems, including the bacterial flagellar motility machine, the injectisome machinery that bacteria use to inject effector proteins into host cells, and probably membrane nanotubes which connect bacterial cells. Here, we accidentally discovered a novel inner membrane-associated complex related to the flagellar T3SS. Examining our lab database, which is comprised of more than 40,000 cryo-tomograms of dozens of species, we discovered that this novel structure is both ubiquitous and ancient, being present in highly divergent classes of bacteria. Discovering a novel, widespread structure related to what are among the best-studied molecular machines in bacteria will open new venues for research aiming at understanding the function and evolution of T3SS proteins.

Activation of the Type III Secretion System of Enteropathogenic Escherichia coli Leads to Remodeling of Its Membrane Composition and Function.

The cell envelope of Gram-negative bacteria is a complex structure, essential for bacterial survival and for resistance to many antibiotics. Channels that cross the bacterial envelope and the host cell membrane form secretion systems that are activated upon attachment to host, enabling bacteria to inject effector molecules into the host cell, required for bacterium-host interaction. The type III secretion system (T3SS) is critical for the virulence of several pathogenic bacteria, including enteropathogenic Escherichia coli (EPEC). EPEC T3SS activation is associated with repression of carbon storage regulator (CsrA), resulting in gene expression remodeling, which is known to affect EPEC central carbon metabolism and contributes to the adaptation to a cell-adherent lifestyle in a poorly understood manner. We reasoned that the changes in the bacterial envelope upon attachment to the host and the activation of a secretion system may involve a modification of the lipid composition of bacterial envelope. Accordingly, we performed a lipidomics analysis on mutant strains that simulate T3SS activation. We saw a shift in glycerophospholipid metabolism toward the formation of lysophospholipids, attributed to corresponding upregulation of the phospholipase gene pldA and the acyltransferase gene ygiH upon T3SS activation in EPEC. We also detected a shift from menaquinones and ubiquinones to undecaprenyl lipids, concomitant with abnormal synthesis of O antigen. The remodeling of lipid metabolism is mediated by CsrA and associated with increased bacterial cell size and zeta potential and a corresponding alteration in EPEC permeability to vancomycin, increasing the sensitivity of T3SS-activated strains and of adherent wild-type EPEC to the antibiotic. IMPORTANCE The characterization of EPEC membrane lipid metabolism upon attachment to the host is an important step toward a better understanding the shift of EPEC, a notable human pathogen, from a planktonic to adherent lifestyle. It may also apply to other pathogenic bacteria that use this secretion system. We predict that upon attachment to host cells, the lipid remodeling upon T3SS activation contributes to bacterial fitness and promotes host colonization, and we show that it is associated with increased cell permeability and higher sensitivity to vancomycin. To the best of our knowledge, this is the first demonstration of a bacterial lipid remodeling due to activation of a secretion system.

Intramuscular mRNA BNT162b2 vaccine against SARS-CoV-2 induces neutralizing salivary IgA.

Intramuscularly administered vaccines stimulate robust serum neutralizing antibodies, yet they are often less competent in eliciting sustainable "sterilizing immunity" at the mucosal level. Our study uncovers a strong temporary neutralizing mucosal component of immunity, emanating from intramuscular administration of an mRNA vaccine. We show that saliva of BNT162b2 vaccinees contains temporary IgA targeting the receptor-binding domain (RBD) of severe acute respiratory syndrome coronavirus-2 spike protein and demonstrate that these IgAs mediate neutralization. RBD-targeting IgAs were found to associate with the secretory component, indicating their bona fide transcytotic origin and their polymeric multivalent nature. The mechanistic understanding of the high neutralizing activity provided by mucosal IgA, acting at the first line of defense, will advance vaccination design and surveillance principles and may point to novel treatment approaches and new routes of vaccine administration and boosting.

Mitochondrial Targeting of the Enteropathogenic Escherichia coli Map Triggers Calcium Mobilization, ADAM10-MAP Kinase Signaling, and Host Cell Apoptosis.

The ability of diarrheagenic bacterial pathogens, such as enteropathogenic Escherichia coli (EPEC), to modulate the activity of mitogen-activated protein kinases (MAPKs) and cell survival has been suggested to benefit bacterial colonization and infection. However, our understanding of the mechanisms by which EPEC modulate these functions is incomplete. In this study, we show that the EPEC type III secreted effector Map stimulates the sheddase activity of the disintegrin and metalloproteinase domain-containing protein 10 (ADAM10) and the ERK and p38 MAPK signaling cascades. Remarkably, all these activities were dependent upon the ability of Map to target host mitochondria, mainly via its mitochondrial toxicity region (MTR). Map targeting of mitochondria disrupted the mitochondrial membrane potential, causing extrusion of mitochondrial Ca2+ into the host cell cytoplasm. We also found that Map targeting of mitochondria is essential for triggering host cell apoptosis. Based on these findings, we propose a model whereby Map imported into mitochondria causes mitochondrial dysfunction and Ca2+ efflux into the host cytoplasm. Since Ca2+ has been reported to promote ADAM10 activation, the acute elevation of Ca2+ in the cytoplasm may stimulate the ADAM10 sheddase activity, resulting in the release of epidermal growth factors that stimulate the ERK signaling cascade. As p38 activity is also Ca2+ sensitive, elevation in cytoplasmic Ca2+ may independently also activate p38. We hypothesize that Map-dependent MAPK activation, combined with Map-mediated mitochondrial dysfunction, evokes mitochondrial host cell apoptosis, potentially contributing to EPEC colonization and infection of the gut.IMPORTANCE Enteropathogenic E. coli (EPEC) is an important human diarrhea-causing bacterium. The pathogenic effects of EPEC largely depend upon its ability to inject a series of proteins, termed effectors, into the host cells. One such effector is the mitochondrion-associated protein (Map). Map has been shown to induce actin-rich projections (i.e., filopodia) on the infected cell surface and activate a Rho GTPase enzyme termed Cdc42. Nonetheless, although most injected Map localizes to host mitochondria, its functions in the mitochondria remain unknown. Here, we show that Map targeting of mitochondria stimulates the disruption of mitochondrial membrane potential to induce Ca2+ efflux into the host cytoplasm. The efflux stimulates the activity of a protein termed ADAM10, which induces activation of a mitogen-activated protein kinase cascade leading to host cell apoptosis. As apoptosis plays a central role in host-pathogen interactions, our findings provide novel insights into the functions of mitochondrial Map in promoting the EPEC disease.

Enteropathogenic Escherichia coli remodels host endosomes to promote endocytic turnover and breakdown of surface polarity.

Enteropathogenic E. coli (EPEC) is an extracellular diarrheagenic human pathogen which infects the apical plasma membrane of the small intestinal enterocytes. EPEC utilizes a type III secretion system to translocate bacterial effector proteins into its epithelial hosts. This activity, which subverts numerous signaling and membrane trafficking pathways in the infected cells, is thought to contribute to pathogen virulence. The molecular and cellular mechanisms underlying these events are not well understood. We investigated the mode by which EPEC effectors hijack endosomes to modulate endocytosis, recycling and transcytosis in epithelial host cells. To this end, we developed a flow cytometry-based assay and imaging techniques to track endosomal dynamics and membrane cargo trafficking in the infected cells. We show that type-III secreted components prompt the recruitment of clathrin (clathrin and AP2), early (Rab5a and EEA1) and recycling (Rab4a, Rab11a, Rab11b, FIP2, Myo5b) endocytic machineries to peripheral plasma membrane infection sites. Protein cargoes, e.g. transferrin receptors, ß1 integrins and aquaporins, which exploit the endocytic pathways mediated by these machineries, were also found to be recruited to these sites. Moreover, the endosomes and cargo recruitment to infection sites correlated with an increase in cargo endocytic turnover (i.e. endocytosis and recycling) and transcytosis to the infected plasma membrane. The hijacking of endosomes and associated endocytic activities depended on the translocated EspF and Map effectors in non-polarized epithelial cells, and mostly on EspF in polarized epithelial cells. These data suggest a model whereby EPEC effectors hijack endosomal recycling mechanisms to mislocalize and concentrate host plasma membrane proteins in endosomes and in the apically infected plasma membrane. We hypothesize that these activities contribute to bacterial colonization and virulence.

Pathogenic E. coli Extracts Nutrients from Infected Host Cells Utilizing Injectisome Components.

Microbiota and intestinal epithelium restrict pathogen growth by rapid nutrient consumption. We investigated how pathogens circumvent this obstacle to colonize the host. Utilizing enteropathogenic E. coli (EPEC), we show that host-attached bacteria obtain nutrients from infected host cell in a process we termed host nutrient extraction (HNE). We identified an inner-membrane protein complex, henceforth termed CORE, as necessary and sufficient for HNE. The CORE is a key component of the EPEC injectisome, however, here we show that it supports the formation of an alternative structure, composed of membranous nanotubes, protruding from the EPEC surface to directly contact the host. The injectisome and flagellum are evolutionarily related, both containing conserved COREs. Remarkably, CORE complexes of diverse ancestries, including distant flagellar COREs, could rescue HNE capacity of EPEC lacking its native CORE. Our results support the notion that HNE is a widespread virulence strategy, enabling pathogens to thrive in competitive niches.

A Ubiquitous Platform for Bacterial Nanotube Biogenesis.

We have previously described the existence of membranous nanotubes, bridging adjacent bacteria, facilitating intercellular trafficking of nutrients, cytoplasmic proteins, and even plasmids, yet components enabling their biogenesis remain elusive. Here we reveal the identity of a molecular apparatus providing a platform for nanotube biogenesis. Using Bacillus subtilis (Bs), we demonstrate that conserved components of the flagellar export apparatus (FliO, FliP, FliQ, FliR, FlhB, and FlhA), designated CORE, dually serve for flagellum and nanotube assembly. Mutants lacking CORE genes, but not other flagellar components, are deficient in both nanotube production and the associated intercellular molecular trafficking. In accord, CORE components are located at sites of nanotube emergence. Deleting COREs of distinct species established that CORE-mediated nanotube formation is widespread. Furthermore, exogenous COREs from diverse species could restore nanotube generation and functionality in Bs lacking endogenous CORE. Our results demonstrate that the CORE-derived nanotube is a ubiquitous organelle that facilitates intercellular molecular trade across the bacterial kingdom.

DksA-HapR-RpoS axis regulates haemagglutinin protease production in Vibrio cholerae.

DksA acts as a co-factor for the intracellular small signalling molecule ppGpp during the stringent response. We recently reported that the expression of the haemagglutinin protease (HAP), which is needed for shedding of the cholera pathogen Vibrio cholerae during the late phase of infection, is significantly downregulated in V. cholerae ∆dksA mutant (∆dksAVc) cells. So far, it has been shown that HAP production by V. cholerae cells is critically regulated by HapR and also by RpoS. Here, we provide evidence that V. cholerae DksA (DksAVc) positively regulates HapR at both the transcriptional and post-transcriptional levels. We show that in ∆dksAVc cells the CsrB/C/D sRNAs, required for the maintenance of intracellular levels of hapR transcripts during the stationary growth, are distinctly downregulated. Moreover, the expression of exponential phase regulatory protein Fis, a known negative regulator of HapR, was found to continue even during the stationary phase in ∆dksAVc cells compared to that of wild-type strain, suggesting another layer of complex regulation of HapR by DksAVc. Extensive reporter construct-based and quantitative reverse-transcriptase PCR (qRT-PCR) analyses supported that RpoS is distinctly downregulated at the post-transcriptional/translational levels in stationary phase-grown ∆dksAVc cells. Since HAP expression through HapR and RpoS is stationary phase-specific in V. cholerae, it appears that DksAVc is also a critical stationary phase regulator for fine tuning of the expression of HAP. Moreover, experimental evidence provided in this study clearly supports that DksAVc is sitting at the top of the hierarchy of regulation of expression of HAP in V. cholerae.

Host cell attachment elicits posttranscriptional regulation in infecting enteropathogenic bacteria.

The mechanisms by which pathogens sense the host and respond by remodeling gene expression are poorly understood. Enteropathogenic Escherichia coli (EPEC), the cause of severe intestinal infection, employs a type III secretion system (T3SS) to inject effector proteins into intestinal epithelial cells. These effectors subvert host cell processes to promote bacterial colonization. We show that the T3SS also functions to sense the host cell and to trigger in response posttranscriptional remodeling of gene expression in the bacteria. We further show that upon effector injection, the effector-bound chaperone (CesT), which remains in the EPEC cytoplasm, antagonizes the posttranscriptional regulator CsrA. The CesT-CsrA interaction provokes reprogramming of expression of virulence and metabolic genes. This regulation is likely required for the pathogen's adaptation to life on the epithelium surface.

Genetic and mutational characterization of the small alarmone synthetase gene relV of Vibrio cholerae.

In Vibrio cholerae, the causative agent of cholera, products of three genes, relA, spoT and relV, govern nutritional stress related stringent response (SR). SR in bacteria is critically regulated by two intracellular small molecules, guanosine 3'-diphosphate 5'-triphosphate (pppGpp) and guanosine 3',5'-bis(diphosphate) (ppGpp), collectively called (p)ppGpp or alarmone. Evolution of relV is unique in V. cholerae because other Gram-negative bacteria carry only relA and spoT genes. Recent reports suggest that RelV is needed for pathogenesis. RelV carries a single (p)ppGpp synthetase or RelA-SpoT domain (SYNTH/RSD) and belongs to the small alarmone synthetase (SAS) family of proteins. Here, we report extensive functional characterizations of the relV gene by constructing several deletion and site-directed mutants followed by their controlled expression in (p)ppGpp(0) cells of Escherichia coli or V. cholerae. Substitution analysis indicated that the amino acid residues K107, D129, R132, L150 and E188 of the RSD region of RelV are essential for its activity. While K107, D129 and E188 are highly conserved in RelA and SAS proteins, L150 appears to be conserved in the latter group of enzymes, and the R132 residue was found to be unique in RelV. Extensive progressive deletion analysis indicated that the amino acid residues at positions 59 and 248 of the RelV protein are the functional N- and C-terminal boundaries, respectively. Since the minimal functional length of RelV was found to be 189 aa, which includes the 94 aa long RSD region, it seems that the flanking residues of the RSD are also important for maintaining the (p)ppGpp synthetase activity.

Functional characterization of the stringent response regulatory gene dksA of Vibrio cholerae and its role in modulation of virulence phenotypes.

In bacteria, nutrient deprivation evokes the stringent response, which is mediated by the small intracellular signaling molecule ppGpp. In Gram negatives, the RelA enzyme synthesizes and SpoT hydrolyzes ppGpp, although the latter protein also has weak synthetase activity. DksA, a recently identified RNA polymerase binding transcription factor, acts as a coregulator along with ppGpp for controlling the stringent response. Recently, we have shown that three genes, relA, spoT, and relV, govern cellular levels of ppGpp during various starvation stresses in the Gram-negative cholera pathogen Vibrio cholerae. Here we report functional characterization of the dksA gene of V. cholerae (dksA(Vc)), coding for the protein DksA(Vc). Extensive genetic analyses of the ΔdksA(Vc) mutants suggest that DksA(Vc) is an important component involved in the stringent response in V. cholerae. Further analysis of mutants revealed that DksA(Vc) positively regulates various virulence-related processes, namely, motility, expression of the major secretory protease, called hemagglutinin protease (HAP), and production of cholera toxin (CT), under in vitro conditions. We found that DksA(Vc) upregulates expression of the sigma factor FliA (σ(28)), a critical regulator of motility in V. cholerae. Altogether, it appears that apart from stringent-response regulation, DksA(Vc) also has important roles in fine regulation of virulence-related phenotypes of V. cholerae.

Genetic components of stringent response in Vibrio cholerae.

Nutritional stress elicits stringent response in bacteria involving modulation of expression of several genes. This is mainly triggered by the intracellular accumulation of two small molecules, namely, guanosine 3'-diphosphate 5'-triphosphate and guanosine 3',5'-bis(diphosphate), collectively called (p)ppGpp. Like in other Gram-negative bacteria, the cellular level of (p)ppGpp is maintained in Vibrio cholerae, the causative bacterial pathogen of the disease cholera, by the products of two genes relA and spoT. However, apart from relA and spoT, a novel gene relV has recently been identified in V. cholerae, the product of which has been shown to be involved in (p)ppGpp synthesis under glucose or fatty acid starvation in a ∆relA ∆spoT mutant background. Furthermore, the GTP binding essential protein CgtA and a non-DNA binding transcription factor DksA also seem to play several important roles in modulating stringent response and regulation of other genes in this pathogen. The present review briefly discusses about the role of all these genes mainly in the management of stringent response in V. cholerae.

Stringent response in Vibrio cholerae: genetic analysis of spoT gene function and identification of a novel (p)ppGpp synthetase gene.

RelA and SpoT of Gram-negative organisms critically regulate cellular levels of (p)ppGpp. Here, we have dissected the spoT gene function of the cholera pathogen Vibrio cholerae by extensive genetic analysis. Unlike Escherichia coli, V. choleraeDeltarelADeltaspoT cells accumulated (p)ppGpp upon fatty acid or glucose starvation. The result strongly suggests RelA-SpoT-independent (p)ppGpp synthesis in V. cholerae. By repeated subculturing of a V. choleraeDeltarelADeltaspoT mutant, a suppressor strain with (p)ppGpp(0) phenotype was isolated. Bioinformatics analysis of V. cholerae whole genome sequence allowed identification of a hypothetical gene (VC1224), which codes for a small protein (approximately 29 kDa) with a (p)ppGpp synthetase domain and the gene is highly conserved in vibrios; hence it has been named relV. Using E. coliDeltarelA or DeltarelADeltaspoT mutant we showed that relV indeed codes for a novel (p)ppGpp synthetase. Further analysis indicated that relV gene of the suppressor strain carries a point mutation at nucleotide position 676 of its coding region (DeltarelADeltaspoT relV676), which seems to be responsible for the (p)ppGpp(0) phenotype. Analysis of a V. choleraeDeltarelADeltaspoTDeltarelV triple mutant confirmed that apart from canonical relA and spoT genes, relV is a novel gene in V. cholerae responsible for (p)ppGpp synthesis.