A bacterial secreted translocator hijacks riboregulators to control type III secretion in response to host cell contact.

Numerous Gram-negative pathogens use a Type III Secretion System (T3SS) to promote virulence by injecting effector proteins into targeted host cells, which subvert host cell processes. Expression of T3SS and the effectors is triggered upon host cell contact, but the underlying mechanism is poorly understood. Here, we report a novel strategy of Yersinia pseudotuberculosis in which this pathogen uses a secreted T3SS translocator protein (YopD) to control global RNA regulators.

Characterization of the Ruler Protein Interaction Interface on the Substrate Specificity Switch Protein in the Type III Secretion System.

Many pathogenic Gram-negative bacteria use the type III secretion system (T3SS) to deliver effector proteins into eukaryotic host cells. In , the switch to secretion of effector proteins is induced first after intimate contact between the bacterium and its eukaryotic target cell has been established, and the T3SS proteins YscP and YscU play a central role in this process. Here we identify the molecular details of the YscP binding site on YscU by means of nuclear magnetic resonance (NMR) spectroscopy.

Increased plasmid copy number is essential for Yersinia T3SS function and virulence.

Pathogenic bacteria have evolved numerous virulence mechanisms that are essential for establishing infections. The enterobacterium Yersinia uses a type III secretion system (T3SS) encoded by a 70-kilobase, low-copy, IncFII-class virulence plasmid. We report a novel virulence strategy in Y.

YscU/FlhB of Yersinia pseudotuberculosis Harbors a C-terminal Type III Secretion Signal.

All type III secretion systems (T3SS) harbor a member of the YscU/FlhB family of proteins that is characterized by an auto-proteolytic process that occurs at a conserved cytoplasmic NPTH motif. We have previously demonstrated that YscUCC, the C-terminal peptide generated by auto-proteolysis of Yersinia pseudotuberculosis YscU, is secreted by the T3SS when bacteria are grown in Ca(2+)-depleted medium at 37 °C. Here, we investigated the secretion of this early T3S-substrate and showed that YscUCC encompasses a specific C-terminal T3S signal within the 15 last residues (U15).

A 2-pyridone-amide inhibitor targets the glucose metabolism pathway of Chlamydia trachomatis.

Unlabelled: In a screen for compounds that inhibit infectivity of the obligate intracellular pathogen Chlamydia trachomatis, we identified the 2-pyridone amide KSK120. A fluorescent KSK120 analogue was synthesized and observed to be associated with the C. trachomatis surface, suggesting that its target is bacterial.

Negatively charged lipid membranes promote a disorder-order transition in the Yersinia YscU protein.

The inner membrane of Gram-negative bacteria is negatively charged, rendering positively charged cytoplasmic proteins in close proximity likely candidates for protein-membrane interactions. YscU is a Yersinia pseudotuberculosis type III secretion system protein crucial for bacterial pathogenesis. The protein contains a highly conserved positively charged linker sequence that separates membrane-spanning and cytoplasmic (YscUC) domains.

Mutations in hemG mediate resistance to salicylidene acylhydrazides, demonstrating a novel link between protoporphyrinogen oxidase (HemG) and Chlamydia trachomatis infectivity.

Salicylidene acylhydrazides (SAHs) inhibit the type III secretion system (T3S) of Yersinia and other Gram-negative bacteria. In addition, SAHs restrict the growth and development of Chlamydia species. However, since the inhibition of Chlamydia growth by SAH is suppressed by the addition of excess iron and since SAHs have an iron-chelating capacity, their role as specific T3S inhibitors is unclear.

Metabolic profiling for detection of Staphylococcus aureus infection and antibiotic resistance.

Due to slow diagnostics, physicians must optimize antibiotic therapies based on clinical evaluation of patients without specific information on causative bacteria. We have investigated metabolomic analysis of blood for the detection of acute bacterial infection and early differentiation between ineffective and effective antibiotic treatment. A vital and timely therapeutic difficulty was thereby addressed: the ability to rapidly detect treatment failures because of antibiotic-resistant bacteria.

Type III secretion translocon assemblies that attenuate Yersinia virulence.

Type III secretion enables bacteria to intoxicate eukaryotic cells with anti-host effectors. A class of secreted cargo are the two hydrophobic translocators that form a translocon pore in the host cell plasma membrane through which the translocated effectors may gain cellular entry. In pathogenic Yersinia, YopB and YopD shape this translocon pore.

Autoproteolysis and intramolecular dissociation of Yersinia YscU precedes secretion of its C-terminal polypeptide YscU(CC).

Type III secretion system mediated secretion and translocation of Yop-effector proteins across the eukaryotic target cell membrane by pathogenic Yersinia is highly organized and is dependent on a switching event from secretion of early structural substrates to late effector substrates (Yops). Substrate switching can be mimicked in vitro by modulating the calcium levels in the growth medium. YscU that is essential for regulation of this switch undergoes autoproteolysis at a conserved N↑PTH motif, resulting in a 10 kDa C-terminal polypeptide fragment denoted YscU(CC).

Yersinia pseudotuberculosis effector YopJ subverts the Nod2/RICK/TAK1 pathway and activates caspase-1 to induce intestinal barrier dysfunction.

Yersinia pseudotuberculosis is an enteropathogenic bacteria that disrupts the intestinal barrier and invades its host through gut-associated lymphoid tissue and Peyer's patches (PP). We show that the Y. pseudotuberculosis effector YopJ induces intestinal barrier dysfunction by subverting signaling of the innate immune receptor Nod2, a phenotype that can be reversed by pretreating with the Nod2 ligand muramyl-dipeptide.

Concerted actions of a thermo-labile regulator and a unique intergenic RNA thermosensor control Yersinia virulence.

Expression of all Yersinia pathogenicity factors encoded on the virulence plasmid, including the yop effector and the ysc type III secretion genes, is controlled by the transcriptional activator LcrF in response to temperature. Here, we show that a protein- and RNA-dependent hierarchy of thermosensors induce LcrF synthesis at body temperature. Thermally regulated transcription of lcrF is modest and mediated by the thermo-sensitive modulator YmoA, which represses transcription from a single promoter located far upstream of the yscW-lcrF operon at moderate temperatures.

Optimization of a sample preparation method for the metabolomic analysis of clinically relevant bacteria.

Metabolomics, or metabolite profiling, is an approach that is increasingly used to study the metabolism of diverse organisms, elucidate biological processes and/or find characteristic biomarkers of physiological states. Here, we describe the optimization of a method for global metabolomic analysis of bacterial cultures, with the following steps. Cells are grown to log-phase, starting from an overnight culture and bacterial concentrations are monitored by measuring the optical density of the cultures at 600 nm.

The RACK1 signaling scaffold protein selectively interacts with Yersinia pseudotuberculosis virulence function.

Many gram-negative bacteria use type III secretion systems to translocate effector proteins into host cells. These effectors interfere with cellular functions in a highly regulated manner resulting in effects that are beneficial for the bacteria. The pathogen Yersinia can resist phagocytosis by eukaryotic cells by translocating Yop effectors into the target cell cytoplasm.

Translocation of surface-localized effectors in type III secretion.

Pathogenic Yersinia species suppress the host immune response by using a plasmid-encoded type III secretion system (T3SS) to translocate virulence proteins into the cytosol of the target cells. T3SS-dependent protein translocation is believed to occur in one step from the bacterial cytosol to the target-cell cytoplasm through a conduit created by the T3SS upon target cell contact. Here, we report that T3SS substrates on the surface of Yersinia pseudotuberculosis are translocated into target cells.

Alternative endogenous protein processing via an autophagy-dependent pathway compensates for Yersinia-mediated inhibition of endosomal major histocompatibility complex class II antigen presentation.

Extracellular Yersinia pseudotuberculosis employs a type III secretion system (T3SS) for translocating virulence factors (Yersinia outer proteins [Yops]) directly into the cytosol of eukaryotic cells. Recently, we used YopE as a carrier molecule for T3SS-dependent secretion and translocation of listeriolysin O (LLO) from Listeria monocytogenes. We demonstrated that translocation of chimeric YopE/LLO into the cytosol of macrophages by Yersinia results in the induction of a codominant antigen-specific CD4 and CD8 T-cell response in orally immunized mice.

The membrane localization domain is required for intracellular localization and autoregulation of YopE in Yersinia pseudotuberculosis.

Recent work has shown that a domain of YopE of Yersinia pseudotuberculosis ranging from amino acids 54 to 75 (R. Krall, Y. Zhang, and J.

Autoproteolysis of YscU of Yersinia pseudotuberculosis is important for regulation of expression and secretion of Yop proteins.

YscU of Yersinia can be autoproteolysed to generate a 10-kDa C-terminal polypeptide designated YscU(CC). Autoproteolysis occurs at the conserved N downward arrowPTH motif of YscU. The specific in-cis-generated point mutants N263A and P264A were found to be defective in proteolysis.

Nod2 mediates susceptibility to Yersinia pseudotuberculosis in mice.

Nucleotide oligomerisation domain 2 (NOD2) is a component of the innate immunity known to be involved in the homeostasis of Peyer patches (PPs) in mice. However, little is known about its role during gut infection in vivo. Yersinia pseudotuberculosis is an enteropathogen causing gastroenteritis, adenolymphitis and septicaemia which is able to invade its host through PPs.

The influence of in vitro fitness defects on pneumococcal ability to colonize and to cause invasive disease.

Background: Streptococcus pneumoniae is a genetically diverse major human pathogen, yet a common colonizer of the nasopharynx. Here we analyzed the influence of defects affecting in vitro growth rate, on the ability of S. pneumoniae to colonize and to cause invasive disease in vivo.

Regulation of Yersinia Yop-effector delivery by translocated YopE.

The bacterial pathogen Yersinia pseudotuberculosis uses a type III secretion (T3S) system to translocate Yop effectors into eukaryotic cells. Effectors are thought to gain access to the cytosol via pores formed in the host cell plasma membrane. Translocated YopE can modulate this pore formation through its GTPase-activating protein (GAP) activity.

Salicylidene acylhydrazides that affect type III protein secretion in Salmonella enterica serovar typhimurium.

A collection of nine salicylidene acylhydrazide compounds were tested for their ability to inhibit the activity of virulence-associated type III secretion systems (T3SSs) in Salmonella enterica serovar Typhimurium. The compounds strongly affected Salmonella pathogenicity island 1 (SPI1) T3SS-mediated invasion of epithelial cells and in vitro secretion of SPI1 invasion-associated effector proteins. The use of a SPI1 effector beta-lactamase fusion protein implicated intracellular entrapment of the protein construct upon application of a salicylidene acylhydrazide, whereas the use of chromosomal transcriptional gene fusions revealed a compound-mediated transcriptional silencing of SPI1.