Injectisome Assembly Research Articles

Enhanced protein translocation to mammalian cells by expression of EtgA transglycosylase in a synthetic injector E. coli strain

BackgroundBacterial type III secretion systems (T3SSs) assemble a multiprotein complex termed the injectisome, which acts as a molecular syringe for translocation of specific effector proteins into the cytoplasm of host cells. The use of injectisomes for delivery of therapeutic proteins into mammalian cells is attractive for biomedical applications. With that aim, we previously generated a non-pathogenic Escherichia coli strain, called Synthetic Injector E. coli (SIEC), which assembles functional injectisomes from enteropathogenic E. coli (EPEC). The assembly of injectisomes in EPEC is assisted by the lytic transglycosylase EtgA, which degrades the peptidoglycan layer. As SIEC lacks EtgA, we investigated whether expression of this transglycosylase enhances the protein translocation capacity of the engineered bacterium.ResultsThe etgA gene from EPEC was integrated into the SIEC chromosome under the control of the inducible tac promoter, generating the strain SIEC-eEtgA. The controlled expression of EtgA had no effect on the growth or viability of bacteria. Upon induction, injectisome assembly was ~ 30% greater in SIEC-eEtgA than in the parental strain, as determined by the level of T3SS translocon proteins, the hemolytic activity of the bacterial strain, and the impairment in flagellar motility. The functionality of SIEC-eEtgA injectisomes was evaluated in a derivative strain carrying a synthetic operon (eLEE5), which was capable of delivering Tir effector protein into the cytoplasm of HeLa cells triggering F-actin polymerization beneath the attached bacterium. Lastly, using β-lactamase as a reporter of T3SS-protein injection, we determined that the protein translocation capacity was ~ 65% higher in the SIEC-EtgA strain than in the parental SIEC strain.ConclusionsWe demonstrate that EtgA enhances the assembly of functional injectisomes in a synthetic injector E. coli strain, enabling the translocation of greater amounts of proteins into the cytoplasm of mammalian cells. Accordingly, EtgA expression may boost the protein translocation of SIEC strains programmed as living biotherapeutics.

Deciphering the interaction network of cytosolic type 3 secretion system proteins in living Yersinia enterocolitica

The virulence-associated type III secretion system (T3SS), a macromolecular protein complex also known as the injectisome, is utilized by gram-negative, pathogenic bacteria to inject species-specific effector proteins into eukaryotic host cells. The cytosolic complex proteins (CCPs), which comprise the cytosolic portion of the injectisome, are highly dynamic and exist in both freely diffusing cytosolic as well as injectisome-bound non-diffusing states. A coordinated network of cytosolic interactions among the CCPs is thought to play a key role in the functional regulation of type 3 secretion by controlling the selection of secreted substrates during injectisome assembly and effector protein secretion.

High-resolution view of the type III secretion export apparatus in situ reveals membrane remodeling and a secretion pathway

Type III protein secretion systems are essential virulence factors for many important pathogenic bacteria. The entire protein secretion machine is composed of several substructures that organize into a holostructure or injectisome. The core component of the injectisome is the needle complex, which houses the export apparatus that serves as a gate for the passage of the secreted proteins through the bacterial inner membrane. Here, we describe a high-resolution structure of the export apparatus of the Salmonella type III secretion system in association with the needle complex and the underlying bacterial membrane, both in isolation and in situ. We show the precise location of the core export apparatus components within the injectisome and bacterial envelope and demonstrate that their deployment results in major membrane remodeling and thinning, which may be central for the protein translocation process. We also show that InvA, a critical export apparatus component, forms a multiring cytoplasmic conduit that provides a pathway for the type III secretion substrates to reach the entrance of the export gate. Combined with structure-guided mutagenesis, our studies provide major insight into potential mechanisms of protein translocation and injectisome assembly.

An Interaction between the Inner Rod Protein YscI and the Needle Protein YscF Is Required to Assemble the Needle Structure of the Yersinia Type Three Secretion System

The type III secretion system is a highly conserved virulence mechanism that is widely distributed in Gram-negative bacteria. It has a syringe-like structure composed of a multi-ring basal body that spans the bacterial envelope and a projecting needle that delivers virulence effectors into host cells. Here, we showed that the Yersinia inner rod protein YscI directly interacts with the needle protein YscF inside the bacterial cells and that this interaction depends on amino acid residues 83-102 in the carboxyl terminus of YscI. Alanine substitution of Trp-85 or Ser-86 abrogated the binding of YscI to YscF as well as needle assembly and the secretion of effectors (Yops) and the needle tip protein LcrV. However, yscI null mutants that were trans-complemented with YscI mutants that bind YscF still assembled the needle and secreted Yops, demonstrating that a direct interaction between YscF and YscI is critical for these processes. Consistently, YscI mutants that did not bind YscF resulted in greatly decreased HeLa cell cytotoxicity. Together, these results show that YscI participates in needle assembly by directly interacting with YscF.

LcrV Mutants That Abolish Yersinia Type III Injectisome Function

LcrV, the type III needle cap protein of pathogenic Yersinia, has been proposed to function as a tether between YscF, the needle protein, and YopB-YopD to constitute the injectisome, a conduit for the translocation of effector proteins into host cells. Further, insertion of LcrV-capped needles from a calcium-rich environment into host cells may trigger the low-calcium signal for effector translocation. Here, we used a genetic approach to test the hypothesis that the needle cap responds to the low-calcium signal by promoting injectisome assembly. Growth restriction of Yersinia pestis in the absence of calcium (low-calcium response [LCR(+)] phenotype) was exploited to isolate dominant negative lcrV alleles with missense mutations in its amber stop codon (lcrV(*327)). The addition of at least four amino acids or the eight-residue Strep tag to the C terminus was sufficient to generate an LCR(-) phenotype, with variant LcrV capping type III needles that cannot assemble the YopD injectisome component. The C-terminal Strep tag appears buried within the cap structure, blocking effector transport even in Y. pestis yscF variants that are otherwise calcium blind, a constitutive type III secretion phenotype. Thus, LcrV(*327) mutants arrest the needle cap in a state in which it cannot respond to the low-calcium signal with either injectisome assembly or the activation of type III secretion. Insertion of the Strep tag at other positions of LcrV produced variants with wild-type LCR(+), LCR(-), or dominant negative LCR(-) phenotypes, thereby allowing us to identify discrete sites within LcrV as essential for its attributes as a secretion substrate, needle cap, and injectisome assembly factor.

Structure of the Type III Secretion Recognition Protein YscU from Yersinia enterocolitica

The inner-membrane protein YscU has an important role during the assembly of the Yersinia enterocolitica type III secretion injectisome. Its cytoplasmic domain (YscU C) recognizes translocators as individual substrates in the export hierarchy. Activation of YscU entails autocleavage at a conserved NPTH motif. Modification of this motif markedly changes the properties of YscU, including translocator export cessation and production of longer injectisome needles. We determined the crystal structures of the uncleaved variants N263A and N263D of YscU C at 2.05 Å and 1.55 Å resolution, respectively. The globular domain is found to consist of a central, mixed β-sheet surrounded by α-helices. The NPTH motif forms a type II β-turn connecting two β-strands. NMR analysis of cleaved and uncleaved YscU C indicates that the global structure of the protein is retained in cleaved YscU C. The structure of YscU C variant N263D reveals that wild type YscU C is poised for cleavage due to an optimal reaction geometry for nucleophilic attack of the scissile bond by the side chain of Asn263. In vivo analysis of N263Q and H266A/R314A YscU variants showed a phenotype that combines the absence of translocator secretion with normal needle-length control. Comparing the structure of YscU to those of related proteins reveals that the linker domain between the N-terminal transmembrane domain and the autocleavage domain can switch from an extended to a largely α-helical conformation, allowing for optimal positioning of the autocleavage domain during injectisome assembly.