Olivera Francetic

Expression of the endogenous type II secretion pathway in Escherichia coli leads to chitinase secretion

Escherichia coli K‐12, the most widely used laboratory bacterium, does not secrete proteins into the extracellular medium under standard growth conditions, despite possessing chromosomal genes encoding a putative type II secretion machinery (secreton). We show that in wild‐type E.coli K‐12, divergent transcription of the two operons in the main chromosomal gsp locus, encoding the majority of the secreton components, is silenced by the nucleoid‐structuring protein H‐NS. In mutants lacking H‐NS, the secreton genes cloned on a moderate‐copy‐number plasmid are expressed and promote efficient secretion of the endogenous, co‐regulated endochitinase ChiA. This is the first time that secretion of an endogenous extracellular protein has been demonstrated in E.coli K‐12.

RECENT PROGRESS AND FUTURE DIRECTIONS IN STUDIES OF THE MAIN TERMINAL BRANCH OF THE GENERAL SECRETORY PATHWAY IN GRAM-NEGATIVE BACTERIA : A REVIEW

The main terminal branch (MTB) of the general secretory pathway is used by a wide variety of Gram- bacteria to transport exoproteins from the periplasm to the outside milieu. Recent work has led to the identification of the function of two of its 14 (or more) components: an enzyme with type-IV prepilin peptidase activity and a chaperone-like protein required for the insertion of another of the MTB components into the outer membrane. Despite these important discoveries, little tangible progress has been made towards identifying MTB components that determine secretion specificity (presumably by binding to cognate exoproteins) or which form the putative channel through which exoproteins are transported across the outer membrane. However, the idea that the single integral outer membrane component of the MTB could line the wall of this channel, and the intriguing possibility that other components of the MTB form a rudimentary type-IV pilus-like structure that might span the periplasm both deserve more careful examination. Although Escherichia coli K-12 does not normally secrete exoproteins, its chromosome contains an apparently complete set of genes coding for MTB components. At least two of these genes code for functional proteins, but the operon in which twelve of the genes are located does not appear to be expressed. We are currently searching for conditions which allow these genes to be expressed with the eventual aim of identifying the protein(s) that E. coli K-12 can secrete.

Intestinal adherence associated with type IV pili of enterohemorrhagic Escherichia coli O157:H7

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 causes hemorrhagic colitis and hemolytic uremic syndrome (HUS) by colonizing the gut mucosa and producing Shiga toxins (Stx). The only factor clearly demonstrated to play a role in EHEC adherence to intestinal epithelial cells is intimin, which binds host cell integrins and nucleolin, as well as a receptor (Tir) that it injects into the host cell. Here we report that EHEC O157:H7 produces adhesive type IV pili, which we term hemorrhagic coli pilus (HCP), composed of a 19-kDa pilin subunit (HcpA) that is encoded by the hcpA chromosomal gene. HCP were observed as bundles of fibers greater than 10 microm in length that formed physical bridges between bacteria adhering to human and bovine host cells. Sera of HUS patients, but not healthy individuals, recognized HcpA, suggesting that the pili are produced in vivo during EHEC infections. Inactivation of the hcpA gene in EHEC EDL933 resulted in significantly reduced adherence to cultured human intestinal and bovine renal epithelial cells and to porcine and bovine gut explants. An escN mutant, which is unable to translocate Tir, adhered less than the hcpA mutant, suggesting that adherence mediated by intimin-Tir interactions is a prelude to HCP-mediated adherence. An hcpA and stx1,2 triple mutant and an hcpA mutant had similar levels of adherence to bovine and human epithelial cells while a stx1,2 double mutant had only a minor defect in adherence, indicating that HCP-mediated adherence and cytotoxicity are independent events. Our data establish that EHEC O157:H7 HCP are intestinal colonization factors that are likely to contribute to the pathogenic potential of this food-borne pathogen.

Type II secretion system: a magic beanstalk or a protein escalator.

Type II protein secretion systems (T2SS) are molecular machines that promote specific transport of folded periplasmic proteins in Gram-negative bacteria, across a dedicated channel in the outer membrane. Secreted substrates, released to the milieu or displayed on the cell surface, contribute to bacterial adaptation to a range of habitats, from deep-sea waters to animal and plant tissues. The past decade has seen remarkable progress in structural, biochemical and functional analysis of T2SS and related systems, bringing new mechanistic insights into these dynamic complexes. This review focuses on recent advances in the field, and discusses open questions regarding the secretion mechanism. This article is part of a Special Issue entitled: Protein trafficking and secretion in bacteria. Guest Editors: Anastassios Economou and Ross Dalbey.

Minor pseudopilin self-assembly primes type II secretion pseudopilus elongation

In Gram‐negative bacteria, type II secretion systems (T2SS) assemble inner membrane proteins of the major pseudopilin PulG (GspG) family into periplasmic filaments, which could drive protein secretion in a piston‐like manner. Three minor pseudopilins PulI, PulJ and PulK are essential for protein secretion in the Klebsiella oxytoca T2SS, but their molecular function is unknown. Here, we demonstrate that together these proteins prime pseudopilus assembly, without actively controlling its length or secretin channel opening. Using molecular dynamics, bacterial two‐hybrid assays, cysteine crosslinking and functional analysis, we show that PulI and PulJ nucleate filament assembly by forming a staggered complex in the plasma membrane. Binding of PulK to this complex results in its partial extraction from the membrane and in a 1‐nm shift between their transmembrane segments, equivalent to the major pseudopilin register in the assembled PulG filament. This promotes fully efficient pseudopilus assembly and protein secretion. Therefore, we propose that PulI, PulJ and PulK self‐assembly is thermodynamically coupled to the initiation of pseudopilus assembly, possibly setting the assembly machinery in motion.

Detailed structural and assembly model of the type II secretion pilus from sparse data.

Many Gram-negative bacteria secrete specific proteins via the type II secretion systems (T2SS). These complex machineries share with the related archaeal flagella and type IV pilus (T4P) biogenesis systems the ability to assemble thin, flexible filaments composed of small, initially inner membrane-localized proteins called “pilins.” In the T2SS from Klebsiella oxytoca, periplasmic pseudopili that are essential for pullulanase (PulA) secretion extend beyond the bacterial surface and form pili when the major pilin PulG is overproduced. Here, we describe the detailed, experimentally validated structure of the PulG pilus generated from crystallographic and electron microscopy data by a molecular modeling approach. Two intermolecular salt bridges crucial for function were demonstrated using single and complementary charge inversions. Double-cysteine substitutions in the transmembrane segment of PulG led to position-specific cross-linking of protomers in assembled pili. These biochemical data provided information on residue distances in the filament that were used to derive a refined model of the T2SS pilus at pseudoatomic resolution. PulG is organized as a right-handed helix of subunits, consistent with protomer organization in gonococcal T4P. The conserved character of residues involved in key hydrophobic and electrostatic interactions within the major pseudopilin family supports the general relevance of this model for T2SS pseudopilus structure.

Signal Recognition Particle-Dependent Inner Membrane Targeting of the PulG Pseudopilin Component of a Type II Secretion System

The pseudopilin PulG is an essential component of the pullulanase-specific type II secretion system from Klebsiella oxytoca. PulG is the major subunit of a short, thin-filament pseudopilus, which presumably elongates and retracts in the periplasm, acting as a dynamic piston to promote pullulanase secretion. It has a signal sequence-like N-terminal segment that, according to studies with green and red fluorescent protein chimeras, anchors unassembled PulG in the inner membrane. We analyzed the early steps of PulG inner membrane targeting and insertion in Escherichia coli derivatives defective in different protein targeting and export factors. The beta-galactosidase activity in strains producing a PulG-LacZ hybrid protein increased substantially when the dsbA, dsbB, or all sec genes tested except secB were compromised by mutations. To facilitate analysis of native PulG membrane insertion, a leader peptidase cleavage site was engineered downstream from the N-terminal transmembrane segment (PrePulG*). Unprocessed PrePulG* was detected in strains carrying mutations in secA, secY, secE, and secD genes, including some novel alleles of secY and secD. Furthermore, depletion of the Ffh component of the signal recognition particle (SRP) completely abolished PrePulG* processing, without affecting the Sec-dependent export of periplasmic MalE and RbsB proteins. Thus, PulG is cotranslationally targeted to the inner membrane Sec translocase by SRP.

Cytoplasmic targeting of IpaC to the bacterial pole directs polar type III secretion in Shigella

Type III secretion (T3S) systems are largely used by pathogenic Gram‐negative bacteria to inject multiple effectors into eukaryotic cells. Upon cell contact, these bacterial microinjection devices insert two T3S substrates into host cell membranes, forming a so‐called ‘translocon’ that is required for targeting of type III effectors in the cell cytosol. Here, we show that secretion of the translocon component IpaC of invasive Shigella occurs at the level of one bacterial pole during cell invasion. Using IpaC fusions with green fluorescent protein variants (IpaCi), we show that the IpaC cytoplasmic pool localizes at an old or new bacterial pole, where secretion occurs upon T3S activation. Deletions in ipaC identified domains implicated in polar localization. Only polar IpaCi derivatives inhibited T3S, while IpaCi fusions with diffuse cytoplasmic localization had no detectable effect on T3S. Moreover, the deletions that abolished polar localization led to secretion defects when introduced in ipaC. These results indicate that cytoplasmic polar localization directs secretion of IpaC at the pole of Shigella, and may represent a mandatory step for T3S.

Towards the Identification of Type II Secretion Signals in a Nonacylated Variant of Pullulanase from Klebsiella oxytoca

Pullulanase (PulA) from the gram-negative bacterium Klebsiella oxytoca is a 116-kDa surface-anchored lipoprotein of the isoamylase family that allows growth on branched maltodextrin polymers. PulA is specifically secreted via a type II secretion system. PelBsp-PulA, a nonacylated variant of PulA made by replacing the lipoprotein signal peptide (sp) with the signal peptide of pectate lyase PelB from Erwinia chrysanthemi, was efficiently secreted into the medium. Two 80-amino-acid regions of PulA, designated A and B, were previously shown to promote secretion of beta-lactamase (BlaM) and endoglucanase CelZ fused to the C terminus. We show that A and B fused to the PelB signal peptide can also promote secretion of BlaM and CelZ but not that of nuclease NucB or several other reporter proteins. However, the deletion of most of region A or all of region B, either individually or together, had only a minor effect on PelBsp-PulA secretion. Four independent linker insertions between amino acids 234 and 324 in PelBsp-PulA abolished secretion. This part of PulA, region C, could contain part of the PulA secretion signal or be important for its correct presentation. Deletion of region C abolished PelBsp-PulA secretion without dramatically affecting its stability. PelBsp-PulA-NucB chimeras were secreted only if the PulA-NucB fusion point was located downstream from region C. The data show that at least three regions of PulA contain information that influences its secretion, depending on their context, and that some reporter proteins might contribute to the secretion of chimeras of which they are a part.

The type II secretion system – a dynamic fiber assembly nanomachine

Type II secretion systems (T2SSs) share common origins and structure with archaeal flagella (archaella) and pili, bacterial competence systems and type IV pili. All of these systems use a conserved ATP-powered machinery to assemble helical fibers that are anchored in the plasma membrane. The T2SSs assemble pseudopili, periplasmic filaments that promote extracellular secretion of folded periplasmic proteins. Comparative analysis of T2SSs and related fiber assembly nanomachines might provide important clues on their functional specificities and dynamics. This review focuses on recent developments in the study of pseudopilus structure and biogenesis, and discusses mechanistic models of pseudopilus function in protein secretion.