Tomoko Kubori

Temporal Regulation of Salmonella Virulence Effector Function by Proteasome-Dependent Protein Degradation

Salmonella enterica invasion of host cells requires the reversible activation of the Rho-family GTPases Cdc42 and Rac1 by the bacterially encoded GEF SopE and the GAP SptP, which exert their function at different times during infection and are delivered into host cells by a type III secretion system. We found that SopE and SptP are delivered in equivalent amounts early during infection. However, SopE is rapidly degraded through a proteosome-mediated pathway, while SptP exhibits much slower degradation kinetics. The half-lives of these effector proteins are determined by their secretion and translocation domains. Chimeric protein analysis indicated that delivery of SptP into host cells by the SopE secretion and translocation domain drastically shortened its half-life. Conversely, delivery of SopE by the SptP secretion and translocation signals significantly increased its half-life, resulting in persistent actin cytoskeleton rearrangements. This regulatory mechanism constitutes a remarkable example of a pathogens adaptation to modulate cellular functions.

Assembly of the inner rod determines needle length in the type III secretion injectisome

Assembly of multi-component supramolecular machines is fundamental to biology, yet in most cases, assembly pathways and their control are poorly understood. An example is the type III secretion machine, which mediates the transfer of bacterial virulence proteins into host cells. A central component of this nanomachine is the needle complex or injectisome, an organelle associated with the bacterial envelope that is composed of a multi-ring base, an inner rod, and a protruding needle. Assembly of this organelle proceeds in sequential steps that require the reprogramming of the secretion machine. Here we provide evidence that, in Salmonella typhimurium, completion of the assembly of the inner rod determines the size of the needle substructure. Assembly of the inner rod, which is regulated by the InvJ protein, triggers conformational changes on the cytoplasmic side of the injectisome, reprogramming the secretion apparatus to stop secretion of the needle protein.

Legionella translocates an E3 ubiquitin ligase that has multiple U‐boxes with distinct functions

Legionella pneumophila has a Dot/Icm type IV secretion system used to translocate a number of ‘effector proteins’ which subvert host cell functions. In this study, we identified 19 novel Dot/Icm substrate proteins using a systematic screening technique. A blast analysis revealed that one of the substrates, which we named LubX (LegionellaU‐box protein), contains two domains that have a remarkable similarity to the U‐box, a domain found in eukaryotic E3 ubiquitin ligases. The expression of LubX is induced upon infection, and most of the LubX produced was translocated into the host cells. LubX has ubiquitin ligase activity in conjunction with UbcH5a or UbcH5c E2 enzymes and mediates polyubiquitination of host Clk1 (Cdc2‐like kinase 1). We demonstrate that one of the U‐boxes (U‐box 1) is critical to the ubiquitin ligation, and the other U‐box (U‐box 2) mediates interaction with Clk1. Thus, the two U‐boxes of LubX have distinct functions, and U‐box 2 plays a non‐canonical role in substrate binding. Although we demonstrate that inhibition of Clk kinase results in a marked reduction of Legionella growth within mouse macrophages, the consequence of Clk1 ubiquitination is still being elucidated. Together, these data suggest that Clk1 is the target host molecule which Legionella modulates during infection.

Genetic Analysis of Assembly of the Salmonella enterica Serovar Typhimurium Type III Secretion-Associated Needle Complex

Several pathogenic bacteria have evolved a specialized protein secretion system termed type III to secrete and deliver effector proteins into eukaryotic host cells. Salmonella enterica serovar Typhimurium uses one such system to mediate entry into nonphagocytic cells. This system is composed of more than 20 proteins which are encoded within a pathogenicity island (SPI-1) located at centisome 63 of its chromosome. A subset of these components form a supramolecular structure, termed the needle complex, that resembles the flagellar hook-basal body complex. The needle complex is composed of a multiple-ring cylindrical base that spans the bacterial envelope and a needle-like extension that protrudes from the bacterial outer surface. Although the components of this structure have been identified, little is known about its assembly. In this study we examined the effect of loss-of-function mutations in each of the type III secretion-associated genes encoded within SPI-1 on the assembly of the needle complex. This analysis indicates that the assembly of this organelle occurs in discrete, genetically separable steps. A model for the assembly pathway of this important organelle is proposed that involves a sec-dependent step leading to the assembly of the base substructure followed by a sec-independent process resulting in the assembly of the needle portion.

Salmonella Type III Secretion-Associated Protein InvE Controls Translocation of Effector Proteins into Host Cells

Salmonella enterica encodes a type III secretion system (TTSS) within a pathogenicity island located at centisome 63 (SPI-1), which is essential for its pathogenicity. This system mediates the transfer of a battery of bacterial proteins into the host cell with the capacity to modulate cellular functions. The transfer process is dependent on the function of protein translocases SipB, SipC, and SipD. We report here that Salmonella protein InvE, which is also encoded within SPI-1, is essential for the translocation of bacterial proteins into host cells. An S. enterica serovar Typhimurium mutant carrying a loss-of-function mutation in invE shows reduced secretion of SipB, SipC, and SipD while exhibiting increased secretion of other TTSS effector proteins. We also demonstrate that InvE interacts with a protein complex formed by SipB, SipC, and their cognate chaperone, SicA. We propose that InvE controls protein translocation by regulating the function of the Sip protein translocases.

Legionella Metaeffector Exploits Host Proteasome to Temporally Regulate Cognate Effector

Pathogen-associated secretion systems translocate numerous effector proteins into eukaryotic host cells to coordinate cellular processes important for infection. Spatiotemporal regulation is therefore important for modulating distinct activities of effectors at different stages of infection. Here we provide the first evidence of “metaeffector,” a designation for an effector protein that regulates the function of another effector within the host cell. Legionella LubX protein functions as an E3 ubiquitin ligase that hijacks the host proteasome to specifically target the bacterial effector protein SidH for degradation. Delayed delivery of LubX to the host cytoplasm leads to the shutdown of SidH within the host cells at later stages of infection. This demonstrates a sophisticated level of coevolution between eukaryotic cells and L. pneumophila involving an effector that functions as a key regulator to temporally coordinate the function of a cognate effector protein.

Type IVB Secretion Systems of Legionella and Other Gram-Negative Bacteria

Type IV secretion systems (T4SSs) play a central role in the pathogenicity of many important pathogens, including Agrobacterium tumefaciens, Helicobacter pylori, and Legionella pneumophila. The T4SSs are related to bacterial conjugation systems, and are classified into two subgroups, type IVA (T4ASS) and type IVB (T4BSS). The T4BSS, which is closely related to conjugation systems of IncI plasmids, was originally found in human pathogen L. pneumophila; pathogenesis by L. pneumophila infection requires functional Dot/Icm T4BSS. A zoonotic pathogen, Coxiella burnetii, and an arthropod pathogen, Rickettsiella grylli – both of which carry T4BSSs highly similar to the Legionella Dot/Icm system – are evolutionarily closely related and comprise a monophyletic group. A growing body of bacterial genomic information now suggests that T4BSSs are not limited to Legionella and related bacteria and IncI plasmids. Here, we review the current knowledge on T4BSS apparatus and component proteins, gained mainly from studies on L. pneumophila Dot/Icm T4BSS. Recent structural studies, along with previous findings, suggest that the Dot/Icm T4BSS contains components with primary or higher-order structures similar to those in other types of secretion systems – types II, III, IVA, and VI, thus highlighting the mosaic nature of T4BSS architecture.

Molecular and functional analysis of the type III secretion signal of the Salmonella enterica InvJ protein

Central to the pathogenicity of Salmonella enterica is the function of a type III secretion system (TTSS) encoded within a pathogenicity island at centisome 63 (SPI‐1). An essential component of this system is a supramolecular structure termed the needle complex. Proteins to be delivered into host cells possess specific signals that route them to the type III secretion pathway. In addition, some bacterial proteins have signals that deliver them to the secretion complex to either become their structural components or exert their function at that location. One of these proteins is InvJ, which controls the length of the needle substructure of the needle complex. In this study, we have analysed the signal that targets InvJ to the TTSS. We found that amino acid residues 4 to 7 of InvJ are necessary and sufficient to mediate secretion of InvJ or a reporter protein in a TTSS‐dependent manner. InvJ secretion was found to be essential for its function in needle length determination, effector protein secretion and bacterial invasion of epithelial cells. Frameshift mutagenesis analysis indicated that the InvJ type III secretion signal sequence tolerates significant alterations in its amino acid sequence without affecting InvJ secretion. Introduction of silent mutations in the secretion signal coding sequence that result in drastically different predicted mRNA folds had no effect on InvJ secretion or expression.

Bacterial flagellation and cell division.

The peritrichous flagella of Salmonella are synthesized and function through many cell generations. There are two different aspects in the relationship between flagellar biogenesis and cell division. Filament growth is independent from the cell cycle and the length of filaments appear to be locally controlled at each flagellar base, whereas the number of filaments (or flagellar basal bodies) is dependent on cell cycle. We present a model to explain how the number of filaments is maintained through generations. We will also introduce a new direction for research that might directly connect flagellation and cell division; the global communication between flagellar genes and external factors of a complex regulatory network in a cell.

Synthesis and Localization of the Salmonella SPI-1 Type III Secretion Needle Complex Proteins PrgI and PrgJ

An essential component of type III secretion systems (TTSS) is a supramolecular structure termed the needle complex. In Salmonella enterica, at least four proteins make up this structure: InvG, PrgH, PrgK, and PrgI. Another protein, PrgJ, is thought to play a role in the assembly of this structure, but its function is poorly understood. We have analyzed the expression and localization of PrgJ and the needle protein PrgI in different S. enterica serovar Typhimurium mutant strains. We found that the levels of PrgI and PrgJ were significantly reduced in a TTSS-deficient invA mutant strain and that the decreased levels were due to protein instability. In addition, we found that PrgJ, although associated with the needle complex in wild-type S. enterica serovar Typhimurium, was absent from needle complexes obtained from an invJ mutant strain, which exhibits very long needle substructures. We suggest that PrgJ is involved in capping the needle substructure of the needle complex.