Targeting early proximal-rod component substrate FlgB to FlhB for flagellar-type III secretion in Salmonella

The Salmonella flagellar secretion apparatus is a member of the type III secretion (T3S) family of export systems in bacteria. After completion of the flagellar motor structure, the hook-basal body (HBB), the flagellar T3S system undergoes a switch from early to late substrate secretion, which results in the expression and assembly of the external, filament propeller-like structure. In order to characterize early substrate secretion-signals in the flagellar T3S system, the FlgB, and FlgC components of the flagellar rod, which acts as the drive-shaft within the HBB, were subject to deletion mutagenesis to identify regions of these proteins that were important for secretion. The β-lactamase protein lacking its Sec-dependent secretion signal (Bla) was fused to the C-terminus of FlgB and FlgC and used as a reporter to select for and quantify the secretion of FlgB and FlgC into the periplasm. Secretion of Bla into the periplasm confers resistance to ampicillin. In-frame deletions of amino acids 9 through 18 and amino acids 39 through 58 of FlgB decreased FlgB secretion levels while deleting amino acid 6 through 14 diminished FlgC secretion levels. Further PCR-directed mutagenesis indicated that amino acid F45 of FlgB was critical for secretion. Single amino acid mutagenesis revealed that all amino acid substitutions at F45 of FlgB position impaired rod assembly, which was due to a defect of FlgB secretion. An equivalent F49 position in FlgC was essential for assembly but not for secretion. This study also revealed that a hydrophobic patch in the cleaved C-terminal domain of FlhB is critical for recognition of FlgB at F45.     

A dominant‐negative needle mutant blocks type III secretion of early but not late substrates in Yersinia

Yersinia pseudotuberculosis uses a type III secretion system (T3SS) to deliver effectors into host cells. A key component of the T3SS is the needle, which is a hollow tube on the bacterial surface through which effectors are secreted, composed of the YscF protein. To study needle assembly, we performed a screen for dominant‐negative yscF alleles that prevented effector secretion in the presence of wild‐type (WT) YscF. One allele, yscF‐L54V, prevents WT YscF secretion and needle assembly, although purified YscF‐L54V polymerizes in vitro. YscF‐L54V binds to its chaperones YscE and YscG, and the YscF‐L54V–EG complex targets to the T3SS ATPase, YscN. We propose that YscF‐L54V stalls at a binding site in the needle assembly pathway following its release from the chaperones, which blocks the secretion of WT YscF and other early substrates required for building a needle. Interestingly, YscF‐L54V does not affect the activity of pre‐assembled actively secreting machines, indicating that a factor and/or binding site required for YscF secretion is absent from T3SS machines already engaged in effector secretion. Thus, substrate switching may involve the removal of an early substrate‐specific binding site as a mechanism to exclude early substrates from Yop‐secreting machines.     

Genetically distinct pathways guide effector export through the type VI secretion system

Bacterial secretion systems often employ molecular chaperones to recognize and facilitate export of their substrates. Recent work demonstrated that a secreted component of the type VI secretion system (T6SS), haemolysin co‐regulated protein (Hcp), binds directly to effectors, enhancing their stability in the bacterial cytoplasm. Herein, we describe a quantitative cellular proteomics screen for T6S substrates that exploits this chaperone‐like quality of Hcp. Application of this approach to the Hcp secretion island I‐encoded T6SS (H1‐T6SS) of Pseudomonas aeruginosa led to the identification of a novel effector protein, termed Tse4 (t ype VI s ecretion e xported 4), subsequently shown to act as a potent intra‐specific H1‐T6SS‐delivered antibacterial toxin. Interestingly, our screen failed to identify two predicted H1‐T6SS effectors, Tse5 and Tse6, which differ from Hcp‐stabilized substrates by the presence of toxin‐associated PAAR‐repeat motifs and genetic linkage to members of the valine‐glycine repeat protein G (vgrG) genes. Genetic studies further distinguished these two groups of effectors: Hcp‐stabilized effectors were found to display redundancy in interbacterial competition with respect to the requirement for the two H1‐T6SS‐exported VgrG proteins, whereas Tse5 and Tse6 delivery strictly required a cognate VgrG. Together, we propose that interaction with either VgrG or Hcp defines distinct pathways for T6S effector export.     

Secretion of atypical protein substrates by the ESAT‐6 Secretion System of Staphylococcus aureus

Staphylococcus aureus encodes the specialized ESAT‐6 Secretion System (ESS). EsxA and EsxB are secreted by the ESS pathway, and share sequence features of ESAT‐6 and CFP‐10 of the Type VII Secretion System (T7SS) of Mycobacterium tuberculosis. Unlike ESAT‐6 and CFP‐10, EsxA and EsxB do not interact. Instead, EsxB associates with a novel substrate, EsxD, and EsxA dimerizes with itself or EsxC (EsaC). Unlike EsxA and EsxB, EsxC and EsxD do not share obvious sequence features of WXG100 proteins nor PE/PPE and Esp families of proteins, all of which belong to the pfam EsxAB clan of mycobacterial T7SS. EsxD carries the C‐terminal motif YxxxD/E that has been proposed to target T7 substrates for secretion in mycobacteria. Here, we find that deletion, but not amino acid substitutions, in this motif prevent secretion of EsxA and EsxC but not EsxB or EsxD. This is unlike the genetic inactivation of esxA, esxB, esxC or esxD that leads to loss of secretion of all four substrates. Thus, substrate secretion can be uncoupled by deleting the last six amino acids of EsxD. The physical association of EsxC and EsxD with canonical WXG100 proteins suggests that these proteins belong to the EsxAB clan.     

Type IX secretion: the generation of bacterial cell surface coatings involved in virulence,gliding motility and the degradation of complex biopolymers

The Type IX secretion system (T9SS) is present in over 1000 sequenced species/strains of the Fibrobacteres‐Chlorobi‐Bacteroidetes superphylum. Proteins secreted by the T9SS have an N‐terminal signal peptide for translocation across the inner membrane via the SEC translocon and a C‐terminal signal for secretion across the outer membrane via the T9SS. Nineteen protein components of the T9SS have been identified including three, SigP, PorX and PorY that are involved in regulation. The inner membrane proteins PorL and PorM and the outer membrane proteins PorK and PorN interact and a complex comprising PorK and PorN forms a large ring structure of 50 nm in diameter. PorU, PorV, PorQ and PorZ form an attachment complex on the cell surface of the oral pathogen, Porphyromonas gingivalis. P. gingivalis T9SS substrates bind to PorV suggesting that after translocation PorV functions as a shuttle protein to deliver T9SS substrates to the attachment complex. The PorU component of the attachment complex is a novel Gram negative sortase which catalyses the cleavage of the C‐terminal signal and conjugation of the protein substrates to lipopolysaccharide, anchoring them to the cell surface. This review presents an overview of the T9SS focusing on the function of T9SS substrates and machinery components.     

New secreted toxins and immunity proteins encoded within the Type VI secretion system gene cluster of Serratia marcescens

Protein secretion systems are critical to bacterial virulence and interactions with other organisms. The Type VI secretion system (T6SS) is found in many bacterial species and is used to target either eukaryotic cells or competitor bacteria. However, T6SS‐secreted proteins have proven surprisingly elusive. Here, we identified two secreted substrates of the antibacterial T6SS from the opportunistic human pathogen, Serratia marcescens. Ssp1 and Ssp2, both encoded within the T6SS gene cluster, were confirmed as antibacterial toxins delivered by the T6SS. Four related proteins encoded around the Ssp proteins (‘Rap’ proteins) included two specifically conferring self‐resistance (‘immunity’) against T6SS‐dependent Ssp1 or Ssp2 toxicity. Biochemical characterization revealed specific, tight binding between cognate Ssp–Rap pairs, forming complexes of 2:2 stoichiometry. The atomic structures of two Rap proteins were solved, revealing a novel helical fold, dependent on a structural disulphide bond, a structural feature consistent with their functional localization. Homologues of the Serratia Ssp and Rap proteins are found encoded together within other T6SS gene clusters, thus they represent founder members of new families of T6SS‐secreted and cognate immunity proteins. We suggest that Ssp proteins are the original substrates of the S. marcescens T6SS, before horizontal acquisition of other T6SS‐secreted toxins. Molecular insight has been provided into how pathogens utilize antibacterial T6SSs to overcome competitors and succeed in polymicrobial niches.     

The extracellular matrix of the oleolytic biofilms of Marinobacter hydrocarbonoclasticus comprises cytoplasmic proteins and T2SS effectors that promote growth on hydrocarbons and lipids

The assimilation of the nearly water insoluble substrates hydrocarbons and lipids by bacteria entails specific adaptations such as the formation of oleolytic biofilms. The present article reports that the extracellular matrix of an oleolytic biofilm formed by Marinobacter hydrocarbonoclasticus at n‐hexadecane–water interfaces is largely composed of proteins typically cytoplasmic such as translation factors and chaperones, and a lesser amount of proteins of unknown function that are predicted extra‐cytoplasmic. Matrix proteins appear to form a structured film on hydrophobic interfaces and were found mandatory for the development of biofilms on lipids, alkanes and polystyrene. Exo‐proteins secreted through the type‐2 secretion system (T2SS) were shown to be essential for the formation of oleolytic biofilms on both alkanes and triglycerides. The T2SS effector involved in biofilm formation on triglycerides was identified as a lipase. In the case of biofilm formation on n‐hexadecane, the T2SS effector is likely involved in the mass transfer, capture or transport of alkanes. We propose that M. hydrocarbonoclasticus uses cytoplasmic proteins released by cell lysis to form a proteinaceous matrix and dedicated proteins secreted through the T2SS to act specifically in the assimilation pathways of hydrophobic substrates.     

Type VI secretion modulates quorum sensing and stress response in Vibrio anguillarum

Type VI protein secretion systems (T6SS) are essential for virulence of several Gram‐negative bacteria. In this study, we identified a T6SS in Vibrio anguillarum, a marine bacterium that causes a hemorrhagic septicemia in fish. A partial operon vtsA‐H (v ibrio t ype s ix secretion) was sequenced and shown to encode eight proteins. VtsE‐H are signature proteins found in other T6SSs, while VtsA‐D are not associated with T6SS studied so far. In‐frame deletions were made in each gene. Secretion of a haemolysin‐co‐regulated‐like protein (Hcp), a protein secreted by all studied T6SSs, was decreased in VtsE‐H. Unexpectedly, VtsA, VtsC and VtsD activated while VtsB and VtsE‐H repressed hcp expression. The T6SS proteins also regulated expression of two extracellular proteases, EmpA and PrtV, but inversely to Hcp expression. This regulation was indirect as T6S positively regulated expression of the stress‐response regulator RpoS and the quorum‐sensing regulator VanT, which positively regulate protease expression. Moreover, VtsA‐H proteins were not needed for virulence but did play a role in various stress responses. Thus, these data characterize a new role for T6S in the ecology of bacteria and we hypothesize this role to be a signal sensing mechanism that modulates the expression of regulators of the general stress response.     

Structural and dynamic properties of bacterial Type IV secretion systems (Review)

The type IV secretion systems (T4SS) are widely distributed among the Gram-negative and –positive bacteria. These systems mediate the transfer of DNA and protein substrates across the cell envelope to bacterial or eukaryotic cells generally through a process requiring direct cell-to-cell contact. Bacteria have evolved T4SS for survival during establishment of pathogenic or symbiotic relationships with eukaryotic hosts. The Agrobacterium tumefaciens VirB/D4 T4SS and related conjugation machines serve as models for detailed mechanistic studies aimed at elucidating the nature of translocation signals, machine assembly pathways and architectures, and the dynamics of substrate translocation. The A. tumefaciens VirB/D4 T4SS are polar-localized organelles composed of a secretion channel and an extracellular T pilus. These T4SS are assembled from 11 or more subunits. whose membrane topologies, intersubunit contacts and, in some cases, 3-dimensional structures are known. Recently, powerful in vivo assays have identified C-terminal translocation signals, defined for the first time the translocation route for a DNA substrate through a type IV secretion channel, and supplied evidence that ATP energy consumption contributes to a late stage of machine morphogenesis. Together, these recent findings describe the mechanics of type IV secretion in unprecedented detail.     

Stretching the arms of the type VI secretion sheath protein

The bacterial type VI secretion system (T6SS) is a multicomponent complex responsible for the translocation of effector proteins into the external milieu. The T6SS consists of an external sheath, an internal rigid tube, a baseplate, and a T6SS‐specific membrane complex. Secretion is accomplished by the contraction of the sheath, which expels the effector‐loaded tube. In this issue of EMBO reports, Brackmann et al 1 show how modifications of the sheath subunits can lock the T6SS assembly in the extended state. These findings allowed Wang et al 2 and Nazarov et al 3 to purify the T6SS sheath–tube–baseplate complex in the extended pre‐secretion state and to analyze its structure using cryo‐electron microscopy (cryoEM).     

Type III export: new uses for an old pathway

Gram-negative bacteria use type III secretion (TTS) systems to translocate proteins into the extracellular environment or directly into eukaryotic cells. These complex secretory systems are assembled from over 20 different structural proteins, including 10 that have counterparts in the flagellar export pathway. Secretion substrates are directed to the TTS machinery via mRNA and/or amino acid secretion signals. TTS chaperones bind to select secretion substrates and assist in the export process. Recent progress in the understanding of TTS is reviewed.     

Identification of potential type III secretion proteins via heterologous expression of Vibrio parahaemolyticus DNA

We employed a heterologous secretion assay to identify proteins potentially secreted by type III secretion systems (T3SSs) in Vibrio parahaemolyticus. N-terminal sequences from 32 proteins within T3SS genomic islands and seven proteins from elsewhere in the chromosome included proteins that were recognized for export by the Yersinia enterocolitica flagellar T3SS.     

Structures of the Shigella flexneri type 3 secretion system protein MxiC reveal conformational variability amongst homologues

Many Gram-negative pathogenic bacteria use a complex macromolecular machine, known as the type 3 secretion system (T3SS), to transfer virulence proteins into host cells. The T3SS is composed of a cytoplasmic bulb, a basal body spanning the inner and outer bacterial membranes, and an extracellular needle. Secretion is regulated by both cytoplasmic and inner membrane proteins that must respond to specific signals in order to ensure that virulence proteins are not secreted before contact with a eukaryotic cell. This negative regulation is mediated, in part, by a family of proteins that are thought to physically block the entrance to the secretion apparatus until an appropriate signal is received following host cell contact. Despite weak sequence homology between proteins of this family, the crystal structures of Shigella flexneri MxiC we present here confirm the conservation of domain topology with the homologue from Yersinia sp. Interestingly, comparison of the Shigella and Yersinia structures reveals a significant structural change that results in substantial domain re-arrangement and opening of one face of the molecule. The conservation of a negatively charged patch on this face suggests it may have a role in binding other components of the T3SS.     

Shigella IpaD has a dual role: signal transduction from the type III secretion system needle tip and intracellular secretion regulation

Type III secretion systems (T3SSs) are protein injection devices essential for the interaction of many Gram‐negative bacteria with eukaryotic cells. While Shigella assembles its T3SS when the environmental conditions are appropriate for invasion, secretion is only activated after physical contact with a host cell. First, the translocators are secreted to form a pore in the host cell membrane, followed by effectors which manipulate the host cell. Secretion activation is tightly controlled by conserved T3SS components: the needle tip proteins IpaD and IpaB, the needle itself and the intracellular gatekeeper protein MxiC. To further characterize the role of IpaD during activation, we combined random mutagenesis with a genetic screen to identify ipaD mutant strains unable to respond to host cell contact. Class II mutants have an overall defect in secretion induction. They map to IpaD's C‐terminal helix and likely affect activation signal generation or transmission. The Class I mutant secretes translocators prematurely and is specifically defective in IpaD secretion upon activation. A phenotypically equivalent mutant was found in mxiC. We show that IpaD and MxiC act in the same intracellular pathway. In summary, we demonstrate that IpaD has a dual role and acts at two distinct locations during secretion activation.     

Chlamydia trachomatis Slc1 is a type III secretion chaperone that enhances the translocation of its invasion effector substrate TARP

Bacterial type III secretion system (T3SS) chaperones pilot substrates to the export apparatus in a secretion‐competent state, and are consequently central to the translocation of effectors into target cells. Chlamydia trachomatis is a genetically intractable obligate intracellular pathogen that utilizes T3SS effectors to trigger its entry into mammalian cells. The only well‐characterized T3SS effector is TARP (translocated actin recruitment protein), but its chaperone is unknown. Here we exploited a known structural signature to screen for putative type III secretion chaperones encoded within the C. trachomatis genome. Using bacterial two‐hybrid, co‐precipitation, cross‐linking and size exclusion chromatography we show that Slc1 (SycE‐like chaperone 1; CT043) specifically interacts with a 200‐amino‐acid residue N‐terminal region of TARP (TARP^1–200). Slc1 formed homodimers in vitro, as shown in cross‐linking and gel filtration experiments. Biochemical analysis of an isolated Slc1–TARP^1–200 complex was consistent with a characteristic 2:1 chaperone–effector stoichiometry. Furthermore, Slc1 was co‐immunoprecipitated with TARP from C. trachomatis elementary bodies. Also, coexpression of Slc1 specifically enhanced host cell translocation of TARP by a heterologous Yersinia enterocolitica T3SS. Taken together, we propose Slc1 as a chaperone of the C. trachomatis T3SS effector TARP.     

Bacterial secretion chaperones

Many Gram-negative bacteria are able to invade hosts by translocation of effectors directly into target cells in processes usually mediated by two very complex secretion systems (SSs), named type III (T3) and type IV (T4) SSs. These syringe-needle injection devices work with intervention of specialized secretion chaperones that, unlike traditional molecular chaperones, do not assist in protein folding and are not energized by ATP. Controversy still surrounds secretion chaperones primary role, but we can say that these chaperones act as: (i) bodyguards to prevent premature aggregation, or as (ii) pilots to direct substrate secretion through the correct secretion system. This family of chaperones does not share primary structure similarity but amazingly equal 3D folds. This mini review has the intent to present updated structural and functional data for several important secretion chaperones, either alone or in complex with their cognate substrates, as well to report on the common features and roles of T3, T4 and flagellar chaperones.