Joseph P. Vogel

KAR2, a karyogamy gene, is the yeast homolog of the mammalian BiP/GRP78 gene

The yeast KAR2 gene was isolated by complementation of a mutation that blocks nuclear fusion. The predicted KAR2 protein sequence is most homologous to mammalian BiP/GRP78 and has several structural features in common with it: a functional secretory signal sequence, a yeast endoplasmic reticulum retention signal (HDEL) at the carboxyl terminus, and the absence of potential N-linked glycosylation sites. Moreover KAR2 is regulated like BiP/GRP78: the level of mRNA is increased by drug treatments and mutations that cause accumulation of secretory precursors in the endoplasmic reticulum. However, unlike BiP/GRP78, KAR2 is also regulated by heat shock. Deletion of the KAR2 gene generated a recessive lethal mutation, showing that BiP/GRP78 function is required for cell viability.

Sec61p and BiP directly facilitate polypeptide translocation into the ER

Secretory proteins are segregated from cytosolic proteins by their translocation into the endoplasmic reticulum (ER). A modified secretory protein trapped during translocation across the ER membrane can be crosslinked to two previously identified proteins, Sec61p and BiP (Kar2p). The dependence of this cross-linking upon proteins and small molecules was examined. Mutations in SEC62 and SEC63 decrease the ability of Sec61p to be cross-linked to the secretory polypeptide trapped in translocation. ATP is also required for interaction of Sec61p with the secretory protein. Three kar2 alleles display defective translocation in vitro. Two of these alleles also decrease the ability of Sec61p to be cross-linked to the secretory protein. The third allele, while exhibiting a severe translocation defect, does not affect the interaction of Sec61p with the secretory protein. These results suggest that Sec61p is directly involved in translocation and that BiP acts at two stages of the translocation cycle.

The Legionella pneumophila LidA protein: a translocated substrate of the Dot/Icm system associated with maintenance of bacterial integrity

Legionella pneumophila establishes a replication vacuole within phagocytes that requires the bacterial Dot/Icm apparatus for its formation. This apparatus is predicted to translocate effectors into host cells. We hypothesized that some translocated proteins also function to maintain the integrity of the Dot/Icm translocator. Mutations that destroy this function are predicted to result in a Dot/Icm complex that poisons the bacterium, resulting in reduced viability. To identify such mutants, strains were isolated (called lid – ) that showed reduced viability on bacteriological medium in the presence of an intact Dot/Icm apparatus, but which had high viability in the absence of the translocator. Several such mutants were analysed in detail to identify candidate strains that may have lost the ability to synthesize a translocated substrate of Dot/Icm. Two such strains had mutations in the lidA gene. The LidA protein exhibits properties expected for a translocated substrate of Dot/Icm that is important for maintenance of bacterial cell integrity: it associates with the phagosomal surface, promotes replication vacuole formation, and is important for both efficient intracellular growth and high viability on bacteriological media after introduction of a plasmid that allows high level expression of the dotA gene.

Type IVB Secretion by Intracellular Pathogens

A growing number of pathogens are being found to possess specialized secretion systems which they use in various ways to subvert host defenses. One class, called type IV, are defined as having homology to the conjugal transfer systems of naturally occurring plasmids. It has been proposed that pathogens with type IV secretion systems have acquired and adapted the conjugal transfer systems of plasmids and now use them to export toxins. Several well‐characterized intracellular pathogens, including Legionella pneumophila, Coxiella burnetii, Brucella abortus, and Rickettsia prowazekii, contain type IV systems which are known or suspected to be of critical importance in their ability to cause disease. Specifically, these systems are believed to be the key factors determining intracellular fate, and thus the ability to replicate and cause disease.

A yeast RNA-binding protein shuttles between the nucleus and the cytoplasm.

RNA-binding proteins have been suggested to move in association with RNA as it leaves the nucleus. The NPL3 gene of the yeast Saccharomyces cerevisiae encodes in nuclear protein with consensus RNA-binding motifs and similarity to heterogeneous nuclear ribonucleoproteins and members of the S/R protein family. We show that although Npl3 is located in the nucleus, it can shuttle between nuclei in yeast heterokaryons. In contrast, other nucleus-targeted proteins do not leave the nucleus under similar conditions. Mutants missing the RNA-binding motifs or the N terminus are still capable of shuttling in and out of the nucleus. Npl3 mutants missing the C terminus fail to localize to the nucleus. Overproduction of Npl3 in wild-type cells shows cell growth. This toxicity depends on the presence of series of unique repeats in the N terminus and localization to the nucleus. We suggest that the properties of Npl3 are consistent with it being involved in export of RNAs from the nucleus.

IcmS-dependent translocation of SdeA into macrophages by the Legionella pneumophila type IV secretion system

Legionella pneumophila replicates inside alveolar macrophages and causes an acute, potentially fatal pneumonia called Legionnaires’ disease. The ability of this bacterium to grow inside of macrophages is dependent on the presence of a functional dot/icm type IV secretion system (T4SS). Proteins secreted by the Dot/Icm T4SS are presumed to alter the host endocytic pathway, allowing L. pneumophila to establish a replicative niche within the host cell. Here we show that a member of the SidE family of proteins interacts with IcmS and is required for full virulence in the protozoan host Acanthamoeba castellanii. Using immunofluorescence microscopy and adenylate cyclase fusions, we show that SdeA is secreted into host cells by L. pneumophila in an IcmS‐dependent manner. The SidE‐like proteins are secreted very early during macrophage infection, suggesting that they are important in the initial formation of the replicative phagosome. Secreted SidE family members show a similar localization to other Dot/Icm substrates, specifically, to the poles of the replicative phagosome. This common localization of secreted substrates of the Dot/Icm system may indicate the formation of a multiprotein complex on the cytoplasmic face of the replicative phagosome.

The Coxiella burnetii Ankyrin Repeat Domain-Containing Protein Family Is Heterogeneous, with C-Terminal Truncations That Influence Dot/Icm-Mediated Secretion

Coxiella burnetii is an obligate intracellular bacterium that directs biogenesis of a parasitophorous vacuole (PV) for replication. Effectors of PV maturation are likely translocated into the host cytosol by a type IV secretion system (T4SS) with homology to the Dot/Icm apparatus of Legionella pneumophila. Since secreted bacterial virulence factors often functionally mimic the activities of host proteins, prokaryotic proteins with eukaryotic features are considered candidate T4SS substrates. Genes encoding proteins with eukaryotic-type ankyrin repeat domains (Anks) were identified upon genome sequencing of the C. burnetii Nine Mile reference isolate, which is associated with a case of human acute Q fever. Interestingly, recent genome sequencing of the G and K isolates, derived from human chronic endocarditis patients, and of the Dugway rodent isolate revealed remarkable heterogeneity in the Ank gene family, with the Dugway isolate harboring the largest number of full-length Ank genes. Using L. pneumophila as a surrogate host, we identified 10 Dugway Anks and 1 Ank specific to the G and K endocarditis isolates translocated into the host cytosol in a Dot/Icm-dependent fashion. A 10-amino-acid C-terminal region appeared to be necessary for translocation, with some Anks also requiring the chaperone IcmS for secretion. Ectopically expressed Anks localized to a variety of subcellular regions in mammalian cells, including microtubules, mitochondria, and the PV membrane. Collectively, these data suggest that C. burnetii isolates translocate distinct subsets of the Ank protein family into the host cytosol, where they modulate diverse functions, some of which may be unique to C. burnetii pathotypes.

Identification of the core transmembrane complex of the Legionella Dot/Icm type IV secretion system

Type IV secretion systems (T4SS) are utilized by a wide range of Gram negative bacteria to deliver protein and DNA substrates to recipient cells. The best characterized T4SS are the type IVA systems, which exhibit extensive similarity to the Agrobacterium VirB T4SS. In contrast, type IVB secretion systems share almost no sequence homology to the type IVA systems, are composed of approximately twice as many proteins, and remain largely uncharacterized. Type IVB systems include the Dot/Icm systems found in the pathogens Legionella and Coxiella and the conjugative apparatus of IncI plasmids. Here we report the first extensive characterization of a type IVB system, the Legionella Dot/Icm secretion apparatus. Based on biochemical and genetic analysis, we discerned the existence of a critical five‐protein subassembly that spans both bacterial membranes and comprises the core of the secretion complex. This transmembrane connection is mediated by protein dimer pairs consisting of two inner membrane proteins, DotF and DotG, which are able to independently associate with DotH/DotC/DotD in the outer membrane. The Legionella core subcomplex appears to be functionally analogous to the Agrobacterium VirB7‐10 subcomplex, suggesting a remarkable conservation of the core subassembly in these evolutionarily distant type IV secretion machines.

The Legionella pneumophila PilT homologue DotB exhibits ATPase activity that is critical for intracellular growth.

The ability of Legionella pneumophila to grow and cause disease in the host is completely dependent on a type IV secretion system known as the Dot/Icm complex. This membrane-spanning apparatus translocates effector molecules into host cells in a process that is poorly understood but that is known to require the putative ATPase DotB. One possible role for DotB is suggested by its similarity to the PilT family of proteins, which mediate pilus retraction. To better understand the molecular behavior of DotB, we have purified the protein and shown that it forms stable homohexameric rings and hydrolyzes ATP with a specific activity of 6.4 nmol of ATP/min/mg of protein. ATPase activity is critical to the function of DotB, as alteration of the conserved Walker box lysine residue resulted in a mutant protein, DotB K162Q, which failed to bind or hydrolyze ATP and which could not complement a DeltadotB strain for intracellular growth in macrophages. Consistent with the ability of DotB to interact with itself, the dotBK162Q allele exhibited transdominance over wild-type dotB, providing the first example of such a mutation in L. pneumophila. Finally, the DotB K162Q mutant protein had a significantly enhanced membrane localization in L. pneumophila compared to wild-type DotB, suggesting a relationship between nucleotide binding and membrane association. These results are consistent with a model in which DotB cycles between the cytoplasm and the Dot/Icm complex at the membrane, where it hydrolyzes nucleotides to provide energy to the complex.

Legionella pneumophila DotU and IcmF Are Required for Stability of the Dot/Icm Complex

ABSTRACT Legionella pneumophila utilizes a type IV secretion system (T4SS) encoded by 26 dot/icm genes to replicate inside host cells and cause disease. In contrast to all other L. pneumophila dot/icm genes, dotU and icmF have homologs in a wide variety of gram-negative bacteria, none of which possess a T4SS. Instead, dotU and icmF orthologs are linked to a locus encoding a conserved cluster of proteins designated IcmF-associated homologous proteins, which has been proposed to constitute a novel cell surface structure. We show here that dotU is partially required for L. pneumophila intracellular growth, similar to the known requirement for icmF. In addition, we show that dotU and icmF are necessary for optimal plasmid transfer and sodium sensitivity, two additional phenotypes associated with a functional Dot/Icm complex. We found that these effects are due to the destabilization of the T4SS at the transition into the stationary phase, the point at which L. pneumophila becomes virulent. Specifically, three Dot proteins (DotH, DotG, and DotF) exhibit decreased stability in a ΔdotU ΔicmF strain. Furthermore, overexpression of just one of these proteins, DotH, is sufficient to suppress the intracellular growth defect of the ΔdotU ΔicmF mutant. This suggests a model where the DotU and IcmF proteins serve to prevent DotH degradation and therefore function to stabilize the L. pneumophila T4SS. Due to their wide distribution among bacterial species and their genetic linkage to known or predicted cell surface structures, we propose that this function in complex stabilization may be broadly conserved.