New structural insights into the bacterial type III secretion system

The virulence-associated type III secretion system (T3SS) enables many Gram-negative bacterial pathogens to translocate proteins into the eukaryotic host cells that they infect. This unique protein transport process is mediated by the type III secretion apparatus (T3SA), a multisubunit membrane-spanning macromolecular assembly comprising >20 different proteins. Recent studies have identified biochemical and structural properties of the core T3SA, in addition to several components constituting this complex, with important implications for both the assembly process and the overall function of the T3SA.     

Transient shielding of intimin and the type III secretion system of enterohemorrhagic and enteropathogenic Escherichia coli by a group 4 capsule

Enterohemorrhagic and enteropathogenic Escherichia coli (EHEC and EPEC, respectively) strains represent a major global health problem. Their virulence is mediated by the concerted activity of an array of virulence factors including toxins, a type III protein secretion system (TTSS), pili, and others. We previously showed that EPEC O127 forms a group 4 capsule (G4C), and in this report we show that EHEC O157 also produces a G4C, whose assembly is dependent on the etp, etk, and wzy genes. We further show that at early time points postinfection, these G4Cs appear to mask surface structures including intimin and the TTSS. This masking inhibited the attachment of EPEC and EHEC to tissue-cultured epithelial cells, diminished their capacity to induce the formation of actin pedestals, and attenuated TTSS-mediated protein translocation into host cells. Importantly, we found that Ler, a positive regulator of intimin and TTSS genes, represses the expression of the capsule-related genes, including etp and etk. Thus, the expression of TTSS and G4C is conversely regulated and capsule production is diminished upon TTSS expression. Indeed, at later time points postinfection, the diminishing capsule no longer interferes with the activities of intimin and the TTSS. Notably, by using the rabbit infant model, we found that the EHEC G4C is required for efficient colonization of the rabbit large intestine. Taken together, our results suggest that temporal expression of the capsule, which is coordinated with that of the TTSS, is required for optimal EHEC colonization of the host intestine.     

The GrlR-GrlA regulatory system coordinately controls the expression of flagellar and LEE-encoded type III protein secretion systems in enterohemorrhagic Escherichia coli

The gene function of the locus of enterocyte effacement (LEE) is essential for full virulence of enterohemorrhagic Escherichia coli (EHEC). Strict control of LEE gene expression is mediated by the coordinated activities of several regulatory elements. We previously reported that the ClpX/ClpP protease positively controls LEE expression by down-regulating intracellular levels of GrlR, a negative regulator of LEE gene expression. We further revealed that the negative effect of GrlR on LEE expression was mediated through GrlA, a positive regulator of LEE expression. In this study, we found that the FliC protein, a major component of flagellar filament, was overproduced in clpXP mutant EHEC, as previously reported for Salmonella. We further found that FliC expression was reduced in a clpXP grlR double mutant. To determine the mediators of this phenotype, FliC protein levels in wild-type, grlR, grlA, and grlR grlA strains were compared. Steady-state levels of FliC protein were reduced only in the grlR mutant, suggesting that positive regulation of FliC expression by GrlR is mediated by GrlA. Correspondingly, cell motility was also reduced in the grlR mutant, but not in the grlA or grlR grlA mutant. Because overexpression of grlA from a multicopy plasmid strongly represses the FliC level, as well as cell motility, we conclude that GrlA acts as a negative regulator of flagellar-gene expression. The fact that an EHEC strain constitutively expressing FlhD/FlhC cannot adhere to HeLa cells leads us to hypothesize that GrlA-dependent repression of the flagellar regulon is important for efficient cell adhesion of EHEC to host cells.     

Hierarchical protein targeting and secretion is controlled by an affinity switch in the type III secretion system of enteropathogenic Escherichia coli

Type III secretion (T3S), a protein export pathway common to Gram‐negative pathogens, comprises a trans‐envelope syringe, the injectisome, with a cytoplasm‐facing translocase channel. Exported substrates are chaperone‐delivered to the translocase, EscV in enteropathogenic Escherichia coli, and cross it in strict hierarchical manner, for example, first “translocators”, then “effectors”. We dissected T3S substrate targeting and hierarchical switching by reconstituting them in vitro using inverted inner membrane vesicles. EscV recruits and conformationally activates the tightly membrane‐associated pseudo‐effector SepL and its chaperone SepD. This renders SepL a high‐affinity receptor for translocator/chaperone pairs, recognizing specific chaperone signals. In a second, SepD‐coupled step, translocators docked on SepL become secreted. During translocator secretion, SepL/SepD suppress effector/chaperone binding to EscV and prevent premature effector secretion. Disengagement of the SepL/SepD switch directs EscV to dedicated effector export. These findings advance molecular understanding of T3S and reveal a novel mechanism for hierarchical trafficking regulation in protein secretion channels.     

SOS regulation of the type III secretion system of enteropathogenic Escherichia coli

Genomes of bacterial pathogens contain and coordinately regulate virulence-associated genes in order to cause disease. Enteropathogenic Escherichia coli (EPEC), a major cause of watery diarrhea in infants and a model gram-negative pathogen, expresses a type III secretion system (TTSS) that is encoded by the locus of enterocyte effacement (LEE) and is necessary for causing attaching and effacing intestinal lesions. Effector proteins encoded by the LEE and in cryptic prophage are injected into the host cell cytoplasm by the TTTS apparatus, ultimately leading to diarrhea. The LEE is comprised of multiple polycistronic operons, most of which are controlled by the global, positive regulator Ler. Here we demonstrated that the LEE2 and LEE3 operons also responded to SOS signaling and that this regulation was LexA dependent. As determined by a DNase I protection assay, purified LexA protein bound in vitro to a predicted SOS box located in the divergent, overlapping LEE2/LEE3 promoters. Expression of the lexA1 allele, encoding an uncleavable LexA protein in EPEC, resulted in reduced secretion, particularly in the absence of the Ler regulator. Finally, we obtained evidence that the cryptic phage-located nleA gene encoding an effector molecule is SOS regulated. Thus, we demonstrated, for the first time to our knowledge, that genes encoding components of a TTSS are regulated by the SOS response, and our data might explain how a subset of EPEC effector proteins, encoded in cryptic prophages, are coordinately regulated with the LEE-encoded TTSS necessary for their translocation into host cells.     

Structural and functional insights into the assembly of type 1 pili from Escherichia coli

Type 1 pili are filamentous protein complexes that are anchored to the outer membrane of uropathogenic Escherichia coli and mediate bacterial adhesion to the surface of urinary epithelium cells. We review here the current status of structural and functional studies on the assembly of type 1 pili.     

Identification of a type III secretion system in uropathogenic Escherichia coli

To determine virulence-related genes in uropathogenic Escherichia coli (UPEC) showing invasiveness to T-24 bladder cancer cells, genomic subtractive hybridization was performed between a highly invasive and a less invasive strain. Forty-nine DNA fragments were isolated from the invasive strain. One of them showed homology with Salmonella invA gene. By chromosomal walking of the strain, a type III secretion system that has been described in E. coli O157:H7 was identified on the genome of the invasive strains. Three strains out of 100 UPEC isolates had a type III secretion system inserted at 64 min of the chromosome, corresponding to E. coli K-12 MG1655. This finding suggested that the type III secretion system could play a part in uropathogenicity of UPEC.     

The rate of folding dictates substrate secretion by the Escherichia coli hemolysin type 1 secretion system

Secretion of the Escherichia coli toxin hemolysin A (HlyA) is catalyzed by the membrane protein complex HlyB-HlyD-TolC and requires a secretion sequence located within the last 60 amino acids of HlyA. The Hly translocator complex exports a variety of passenger proteins when fused N-terminal to this secretion sequence. However, not all fusions are secreted efficiently. Here, we demonstrate that the maltose binding protein (MalE) lacking its natural export signal and fused to the HlyA secretion signal is poorly secreted by the Hly system. We anticipated that folding kinetics might be limiting secretion, and we therefore introduced the "folding" mutation Y283D. Indeed this mutant fusion protein was secreted at a much higher level. This level was further enhanced by the introduction of a second MalE folding mutation (V8G or A276G). Secretion did not require the molecular chaperone SecB. Folding analysis revealed that all mutations reduced the refolding rate of the substrate, whereas the unfolding rate was unaffected. Thus, the efficiency of secretion by the Hly system is dictated by the folding rate of the substrate. Moreover, we demonstrate that fusion proteins defective in export can be engineered for secretion while still retaining function.     

Novel insights into the pathological mechanisms of metabolic related dyslipidemia

Due to the technological advances, it has been well-established that obesity is strongly correlated with various health problems. Among these problems, dyslipidemia is one of the most important concomitant symptoms under obese status which is the main driving force behind the pathological progression of cardio-metabolic disorder diseases. Importantly, the type of dyslipidemia, arising from concerted action of obesity, has been identified as “metabolic related dyslipidemia”, which is characterized by increased circulating levels of Low density lipoprotein cholesterol (LDL-C), Triglycerides (TG) accompanied by lower circulating levels of High density lipoprotein cholesterol (HDL-C). On the other hand, the metabolic related dyslipidemia is being verified as a vital link between obesity and hypertension, diabetes mellitus, and Cardiovascular disease (CVD). In this review, we summarized the current understanding of metabolic related dyslipidemia and the potential mechanisms which lead to the pathogenesis of obesity. Meanwhile, we also summarized the emerging results which focused on several novel lipid bio-markers in metabolic related dyslipidemia, such as pro-protein convertase subtilisin/kexin type 9 (PCSK9) and sphingosine-1-phosphate (S1P), and their potential use as biomarkers of metabolic related dyslipidemia.

     

Type III secretion of EspB in enterohemorrhagic Escherichia coli O157:H7

EspB of enterohemorrhagic Escherichia coli O157:H7 is one of the type III proteins, categorized as translocators, that are secreted in abundance. To define the secretion determinants, different fragments of EspB were fused in recombinant proteins and the proteins secreted into media analyzed by Western blot. The results indicated that the C-terminal 30 residues of EspB were dispensable for secretion whereas the N-terminal first 117 residues played a major role. However, this N-terminal segment alone was not sufficient to confer the secretion. To acquire basic activity, the EspB fusion protein had to contain the N-terminal segment and another segment consisting of either residues 118–190 or residues 191–282. It is possible that the N-terminal region may act as the primary component of the secretion signal while other determinants help to maintain a conformation of EspB favorable for secretion. However, alternative mechanisms cannot be completely excluded. Not withstanding this, the signal for the type III secretion of EspB is apparently distinct from those previously described for the secretion of effector proteins such as Yops in Yersinia.     

An inhibitory mechanism of action of coiled‐coil peptides against type three secretion system from enteropathogenic Escherichia coli

Human pathogenic gram‐negative bacteria, such as enteropathogenic Escherichia coli (EPEC), rely on type III secretion systems (T3SS) to translocate virulence factors directly into host cells. The coiled‐coil domains present in the structural proteins of T3SS are conformed by amphipathic alpha‐helical structures that play an important role in the protein‐protein interaction and are essential for the assembly of the translocation complex. To investigate the inhibitory capacity of these domains on the T3SS of EPEC, we synthesized peptides between 7 and 34 amino acids based on the coiled‐coil domains of proteins that make up this secretion system. This analysis was performed through in vitro hemolysis assays by assessing the reduction of T3SS‐dependent red blood cell lysis in the presence of the synthesized peptides. After confirming its inhibitory capacity, we performed molecular modeling assays using combined techniques, docking‐molecular dynamic simulations, and quantum‐mechanic calculations of the various peptide‐protein complexes, to improve the affinity of the peptides to the target proteins selected from T3SS. These techniques allowed us to demonstrate that the peptides with greater inhibitory activity, directed against the coiled‐coil domain of the C‐terminal region of EspA, present favorable hydrophobic and hydrogen bond molecular interactions. Particularly, the hydrogen bond component is responsible for the stabilization of the peptide‐protein complex. This study demonstrates that compounds targeting T3SS from pathogenic bacteria can indeed inhibit bacterial infection by presenting a higher specificity than broad‐spectrum antibiotics. In turn, these peptides could be taken as initial structures to design and synthesize new compounds that mimic their inhibitory pharmacophoric pattern.     

Structural insights into the catalytic mechanism of Escherichia coli selenophosphate synthetase

Selenophosphate synthetase (SPS) catalyzes the synthesis of selenophosphate, the selenium donor for the biosynthesis of selenocysteine and 2-selenouridine residues in seleno-tRNA. Selenocysteine, known as the 21st amino acid, is then incorporated into proteins during translation to form selenoproteins which serve a variety of cellular processes. SPS activity is dependent on both Mg(2+) and K(+) and uses ATP, selenide, and water to catalyze the formation of AMP, orthophosphate, and selenophosphate. In this reaction, the gamma phosphate of ATP is transferred to the selenide to form selenophosphate, while ADP is hydrolyzed to form orthophosphate and AMP. Most of what is known about the function of SPS has derived from studies investigating Escherichia coli SPS (EcSPS) as a model system. Here we report the crystal structure of the C17S mutant of SPS from E. coli (EcSPS(C17S)) in apo form (without ATP bound). EcSPS(C17S) crystallizes as a homodimer, which was further characterized by analytical ultracentrifugation experiments. The glycine-rich N-terminal region (residues 1 through 47) was found in the open conformation and was mostly ordered in both structures, with a magnesium cofactor bound at the active site of each monomer involving conserved aspartate residues. Mutating these conserved residues (D51, D68, D91, and D227) along with N87, also found at the active site, to alanine completely abolished AMP production in our activity assays, highlighting their essential role for catalysis in EcSPS. Based on the structural and biochemical analysis of EcSPS reported here and using information obtained from similar studies done with SPS orthologs from Aquifex aeolicus and humans, we propose a catalytic mechanism for EcSPS-mediated selenophosphate synthesis.     

Structural insights into the GTPase domain of Escherichia coli MnmE protein

The Escherichia coli MnmE protein is a 50-kDa multidomain GTPase involved in tRNA modification. Its homologues in eukaryotes are crucial for mitochondrial respiration and, thus, it is thought that the human protein might be involved in mitochondrial diseases. Unlike Ras, MnmE shows a high intrinsic GTPase activity and requires effective GTP hydrolysis, and not simply GTP binding, to be functionally active. The isolated MnmE G-domain (165 residues) conserves the GTPase activity of the entire protein, suggesting that it contains the catalytic residues for GTP hydrolysis. To explore the GTP hydrolysis mechanism of MnmE, we analyzed the effect of low pH on binding and hydrolysis of GTP, as well as on the formation of a MnmE transition state mimic. GTP hydrolysis by MnmE, but not GTP binding or formation of a complex with mant-GDP and aluminium fluoride, is impaired at acidic pH, suggesting that the chemistry of the transition state mimic is different to that of the true transition state, and that some residue(s), critical for GTP hydrolysis, is severely affected by low pH. We use a nuclear magnetic resonance (NMR)-based approach to get insights into the MnmE structure and properties. The combined use of NMR restraints and homology structural information allowed the determination of the MnmE G-domain structure in its free form. Chemical shift structure-based prediction provided a good basis for structure refinement and validation. Our data support that MnmE, unlike other GTPases, does not use an arginine finger to drive catalysis, although Arg252 may play a role in stabilization of the transition state.     

EscA is a crucial component of the type III secretion system of enteropathogenic Escherichia coli

The virulence of many Gram-negative pathogens is associated with type III secretion systems (T3SSs), which deliver virulence effector proteins into the cytoplasm of host cells. Components of enteropathogenic Escherichia coli (EPEC) T3SS are encoded within the locus of enterocyte effacement (LEE). While most LEE-encoded T3SS proteins in EPEC have assigned names and functions, a few of them remain poorly characterized. Here, we studied a small LEE-encoded protein, Orf15, that shows no homology to other T3SS/flagellar proteins and is only present in attaching and effacing pathogens, including enterohemorrhagic E. coli and Citrobacter rodentium. Our findings demonstrated that it is essential for type III secretion (T3S) and that it is localized to the periplasm and associated with the inner membrane. Membrane association was driven by the N-terminal 19 amino acid residues, which were also shown to be essential for T3S. Consistent with its localization, Orf15 was found to interact with the EPEC T3SS outer membrane ring component, EscC, which was previously shown to be embedded within the outer membrane and protruding into the periplasmic space. Interestingly, we found that the predicted coiled-coil structure of Orf15 is critical for the protein's function. Overall, our findings suggest that Orf15 is a structural protein that contributes to the structural integrity of the T3S complex, and therefore we propose to rename it EscA.     

Real-time analysis of effector translocation by the type III secretion system of enteropathogenic Escherichia coli

Bacteria use type III secretion systems (TTSS) to translocate effector proteins into host cells. Better understanding of the TTSS and its effectors' functions will require assays to measure their activities in vivo and in real time. We designed a real-time, high-throughput translocation assay that utilizes fusions of effector genes to the beta-lactamase reporter gene, positioned under the effector's native promoter and chromosomal location. Using this assay, we simultaneously and quantitatively analyzed the translocation kinetics of six core enteropathogenic E. coli effectors, EspF, EspG, EspH, EspZ, Map, and Tir. A distinct order in the efficiencies of effector translocation was observed. Translocation efficiency was determined by multiple factors, including the intrabacterial effector concentration, effector-chaperone interactions, the efficiency of bacterial attachment to the host cells, and possibly also by a translocation autoinhibition mechanism. The described real-time translocation assay could be easily adapted for varied applications in the study of bacterial pathogenesis.     

A degenerate type III secretion system from septicemic Escherichia coli contributes to pathogenesis

The type III secretion system (T3SS) is an important virulence factor used by several gram-negative bacteria to deliver effector proteins which subvert host cellular processes. Enterohemorrhagic Escherichia coli O157 has a well-defined T3SS involved in attachment and effacement (ETT1) and critical for virulence. A gene cluster potentially encoding an additional T3SS (ETT2), which resembles the SPI-1 system in Salmonella enterica, was found in its genome sequence. The ETT2 gene cluster has since been found in many E. coli strains, but its in vivo role is not known. Many of the ETT2 gene clusters carry mutations and deletions, raising the possibility that they are not functional. Here we show the existence in septicemic E. coli strains of an ETT2 gene cluster, ETT2(sepsis), which, although degenerate, contributes to pathogenesis. ETT2(sepsis) has several premature stop codons and a large (5 kb) deletion, which is conserved in 11 E. coli strains from cases of septicemia and newborn meningitis. A null mutant constructed to remove genes coding for the putative inner membrane ring of the secretion complex exhibited significantly reduced virulence. These results are the first demonstration of the importance of ETT2 for pathogenesis.