Salmonella exploits host Rho GTPase signalling pathways through the phosphatase activity of SopB.

Salmonella uses Type 3 secretion systems (T3SSs) to deliver virulence factors, called effectors, into host cells during infection. The T3SS effectors promote invasion into host cells and the generation of a replicative niche. SopB is a T3SS effector that plays an important role in Salmonella pathogenesis through its lipid phosphatase activity. Here, we show that SopB mediates the recruitment of Rho GTPases (RhoB, RhoD, RhoH, and RhoJ) to bacterial invasion sites. RhoJ contributes to Salmonella invasion, and RhoB and RhoH play an important role in Akt activation. R-Ras1 also contributes to SopB-dependent Akt activation by promoting the localised production of PI(3,4)P2 /PI(3,4,5)P3 . Our studies reveal new signalling factors involved in SopB-dependent Salmonella infection.

Listeria monocytogenes exploits efferocytosis to promote cell-to-cell spread.

Efferocytosis, the process by which dying or dead cells are removed by phagocytosis, has an important role in development, tissue homeostasis and innate immunity. Efferocytosis is mediated, in part, by receptors that bind to exofacial phosphatidylserine (PS) on cells or cellular debris after loss of plasma membrane asymmetry. Here we show that a bacterial pathogen, Listeria monocytogenes, can exploit efferocytosis to promote cell-to-cell spread during infection. These bacteria can escape the phagosome in host cells by using the pore-forming toxin listeriolysin O (LLO) and two phospholipase C enzymes. Expression of the cell surface protein ActA allows L. monocytogenes to activate host actin regulatory factors and undergo actin-based motility in the cytosol, eventually leading to formation of actin-rich protrusions at the cell surface. Here we show that protrusion formation is associated with plasma membrane damage due to LLO's pore-forming activity. LLO also promotes the release of bacteria-containing protrusions from the host cell, generating membrane-derived vesicles with exofacial PS. The PS-binding receptor TIM-4 (encoded by the Timd4 gene) contributes to efficient cell-to-cell spread by L. monocytogenes in macrophages in vitro and growth of these bacteria is impaired in Timd4(-/-) mice. Thus, L. monocytogenes promotes its dissemination in a host by exploiting efferocytosis. Our results indicate that PS-targeted therapeutics may be useful in the fight against infections by L. monocytogenes and other bacteria that use similar strategies of cell-to-cell spread during infection.

Multiple host kinases contribute to Akt activation during Salmonella infection.

SopB is a type 3 secreted effector with phosphatase activity that Salmonella employs to manipulate host cellular processes, allowing the bacteria to establish their intracellular niche. One important function of SopB is activation of the pro-survival kinase Akt/protein kinase B in the infected host cell. Here, we examine the mechanism of Akt activation by SopB during Salmonella infection. We show that SopB-mediated Akt activation is only partially sensitive to PI3-kinase inhibitors LY294002 and wortmannin in HeLa cells, suggesting that Class I PI3-kinases play only a minor role in this process. However, depletion of PI(3,4) P2/PI(3-5) P3 by expression of the phosphoinositide 3-phosphatase PTEN inhibits Akt activation during Salmonella invasion. Therefore, production of PI(3,4) P2/PI(3-5) P3 appears to be a necessary event for Akt activation by SopB and suggests that non-canonical kinases mediate production of these phosphoinositides during Salmonella infection. We report that Class II PI3-kinase beta isoform, IPMK and other kinases identified from a kinase screen all contribute to Akt activation during Salmonella infection. In addition, the kinases required for SopB-mediated activation of Akt vary depending on the type of infected host cell. Together, our data suggest that Salmonella has evolved to use a single effector, SopB, to manipulate a remarkably large repertoire of host kinases to activate Akt for the purpose of optimizing bacterial replication in its host.

Activation of antibacterial autophagy by NADPH oxidases.

Autophagy plays an important role in immunity to microbial pathogens. The autophagy system can target bacteria in phagosomes, promoting phagosome maturation and preventing pathogen escape into the cytosol. Recently, Toll-like receptor (TLR) signaling from phagosomes was found to initiate their targeting by the autophagy system, but the mechanism by which TLR signaling activates autophagy is unclear. Here we show that autophagy targeting of phagosomes is not exclusive to those containing TLR ligands. Engagement of either TLRs or the Fcgamma receptors (FcgammaRs) during phagocytosis induced recruitment of the autophagy protein LC3 to phagosomes with similar kinetics. Both receptors are known to activate the NOX2 NADPH oxidase, which plays a central role in microbial killing by phagocytes through the generation of reactive oxygen species (ROS). We found that NOX2-generated ROS are necessary for LC3 recruitment to phagosomes. Antibacterial autophagy in human epithelial cells, which do not express NOX2, was also dependent on ROS generation. These data reveal a coupling of oxidative and nonoxidative killing activities of the NOX2 NADPH oxidase in phagocytes through autophagy. Furthermore, our results suggest a general role for members of the NOX family in regulating autophagy.

Listeriolysin O allows Listeria monocytogenes replication in macrophage vacuoles.

Listeria monocytogenes is an intracellular bacterial pathogen that replicates rapidly in the cytosol of host cells during acute infection. Surprisingly, these bacteria were found to occupy vacuoles in liver granuloma macrophages during persistent infection of severe combined immunodeficient (SCID) mice. Here we show that L. monocytogenes can replicate in vacuoles within macrophages. In livers of SCID mice infected for 21 days, we observed bacteria in large LAMP1(+) compartments that we termed spacious Listeria-containing phagosomes (SLAPs). SLAPs were also observed in vitro, and were found to be non-acidic and non-degradative compartments that are generated in an autophagy-dependent manner. The replication rate of bacteria in SLAPs was found to be reduced compared to the rate of those in the cytosol. Listeriolysin O (LLO, encoded by hly), a pore-forming toxin essential for L. monocytogenes virulence, was necessary and sufficient for SLAP formation. A L. monocytogenes mutant with low LLO expression was impaired for phagosome escape but replicated slowly in SLAPs over a 72 h period. Therefore, our studies reveal a role for LLO in promoting L. monocytogenes replication in vacuoles and suggest a mechanism by which this pathogen can establish persistent infection in host macrophages.

Listeria monocytogenes evades killing by autophagy during colonization of host cells.

Listeria monocytogenes is an intracellular pathogen that is able to colonize the cytosol of macrophages. Here we examined the interaction of this pathogen with autophagy, a host cytosolic degradative pathway that constitutes an important component of innate immunity towards microbial invaders. L. monocytogenes infection induced activation of the autophagy system in macrophages. At 1 h post infection (p.i.), a population of intracellular bacteria ( approximately 37%) colocalized with the autophagy marker LC3. These bacteria were within vacuoles and were targeted by autophagy in an LLO-dependent manner. At later stages in infection (by 4 h p.i.), the majority of L. monocytogenes escaped into the cytosol and rapidly replicated. At these times, less than 10% of intracellular bacteria colocalized with LC3. We found that ActA expression was sufficient to prevent autophagy of bacteria in the cytosol of macrophages. Surprisingly, ActA expression was not strictly necessary, indicating that other virulence factors were involved. Accordingly, we also found a role for the bacterial phospholipases, PI-PLC and PC-PLC, in autophagy evasion, as bacteria lacking phospholipase expression were targeted by autophagy at later times in infection. Together, our results demonstrate that L. monocytogenes utilizes multiple mechanisms to avoid destruction by the autophagy system during colonization of macrophages.

ALIS are stress-induced protein storage compartments for substrates of the proteasome and autophagy.

Misfolded proteins can be directed into cytoplasmic aggregates such as aggresomes and dendritic cell aggresome-like induced structures (DALIS). DALIS were originally identified in lipopolysaccharide-stimulated dendritic cells and act as storage compartments for polyubiquitinated Defective Ribosomal Products (DRiPs) prior to their clearance by the proteasome. Here we demonstrate that ubiquitinated protein aggregates that are similar to DALIS, and not related to aggresomes, can be observed in several cell types in response to stress, including oxidative stress, transfection, and starvation. Significantly, both immune and nonimmune cells could form these aggresome-like induced structures (ALIS). Protein synthesis was essential for ALIS formation in response to oxidative stress, indicating that DRiP formation was required. Furthermore, puromycin, which increases DRiP formation, was sufficient to induce ALIS formation. Inhibition of either proteasomes or of autophagy interfered with ALIS clearance in puromycin treated cells. Autophagy inhibition enhanced ALIS formation under a variety of stress conditions. During starvation, ALIS formation in autophagy-deficient cells was only partially inhibited by protein synthesis inhibitors, indicating that both long-lived proteins and DRiPs can be targeted to ALIS. Together, these findings demonstrate that ALIS act as generalized stress-induced protein storage compartments for substrates of the proteasome and autophagy.

Cutting edge: microbial products elicit formation of dendritic cell aggresome-like induced structures in macrophages.

In response to a maturation stimulus, dendritic cells undergo the formation of ubiquitinated protein aggregates known as dendritic cell aggresome-like induced structures (DALIS). DALIS are thought to act as Ag storage structures, allowing for the prioritized degradation of proteins during infection. In this study, we demonstrate that murine macrophages can also form ubiquitinated protein aggregates that are indistinguishable from DALIS. These were formed in a dose- and time-dependent manner, and in response to a variety of microbial products. Surprisingly, the proteasome did not accumulate on these ubiquitinated protein structures, further underlining the difference between DALIS and aggresomes. Our studies suggest that DALIS formation is important for the function of Ag-presenting immune cells during infection.

RPAP1, a novel human RNA polymerase II-associated protein affinity purified with recombinant wild-type and mutated polymerase subunits.

We have programmed human cells to express physiological levels of recombinant RNA polymerase II (RNAPII) subunits carrying tandem affinity purification (TAP) tags. Double-affinity chromatography allowed for the simple and efficient isolation of a complex containing all 12 RNAPII subunits, the general transcription factors TFIIB and TFIIF, the RNAPII phosphatase Fcp1, and a novel 153-kDa polypeptide of unknown function that we named RNAPII-associated protein 1 (RPAP1). The TAP-tagged RNAPII complex is functionally active both in vitro and in vivo. A role for RPAP1 in RNAPII transcription was established by shutting off the synthesis of Ydr527wp, a Saccharomyces cerevisiae protein homologous to RPAP1, and demonstrating that changes in global gene expression were similar to those caused by the loss of the yeast RNAPII subunit Rpb11. We also used TAP-tagged Rpb2 with mutations in fork loop 1 and switch 3, two structural elements located strategically within the active center, to start addressing the roles of these elements in the interaction of the enzyme with the template DNA during the transcription reaction.

High-definition macromolecular composition of yeast RNA-processing complexes.

A remarkably large collection of evolutionarily conserved proteins has been implicated in processing of noncoding RNAs and biogenesis of ribonucleoproteins. To better define the physical and functional relationships among these proteins and their cognate RNAs, we performed 165 highly stringent affinity purifications of known or predicted RNA-related proteins from Saccharomyces cerevisiae. We systematically identified and estimated the relative abundance of stably associated polypeptides and RNA species using a combination of gel densitometry, protein mass spectrometry, and oligonucleotide microarray hybridization. Ninety-two discrete proteins or protein complexes were identified comprising 489 different polypeptides, many associated with one or more specific RNA molecules. Some of the pre-rRNA-processing complexes that were obtained are discrete sub-complexes of those previously described. Among these, we identified the IPI complex required for proper processing of the ITS2 region of the ribosomal RNA primary transcript. This study provides a high-resolution overview of the modular topology of noncoding RNA-processing machinery.

A Snf2 family ATPase complex required for recruitment of the histone H2A variant Htz1.

Deletions of three yeast genes, SET2, CDC73, and DST1, involved in transcriptional elongation and/or chromatin metabolism were used in conjunction with genetic array technology to screen approximately 4700 yeast deletions and identify double deletion mutants that produce synthetic growth defects. Of the five deletions interacting genetically with all three starting mutations, one encoded the histone H2A variant Htz1 and three encoded components of a novel 13 protein complex, SWR-C, containing the Snf2 family ATPase, Swr1. The SWR-C also copurified with Htz1 and Bdf1, a TFIID-interacting protein that recognizes acetylated histone tails. Deletions of the genes encoding Htz1 and seven nonessential SWR-C components caused a similar spectrum of synthetic growth defects when combined with deletions of 384 genes involved in transcription, suggesting that Htz1 and SWR-C belong to the same pathway. We show that recruitment of Htz1 to chromatin requires the SWR-C. Moreover, like Htz1 and Bdf1, the SWR-C promotes gene expression near silent heterochromatin.

Organization and function of APT, a subcomplex of the yeast cleavage and polyadenylation factor involved in the formation of mRNA and small nucleolar RNA 3'-ends.

Messenger RNA 3'-end formation is functionally coupled to transcription by RNA polymerase II. By tagging and purifying Ref2, a non-essential protein previously implicated in mRNA cleavage and termination, we isolated a multiprotein complex, holo-CPF, containing the yeast cleavage and polyadenylation factor (CPF) and six additional polypeptides. The latter can form a distinct complex, APT, in which Pti1, Swd2, a type I protein phosphatase (Glc7), Ssu72 (a TFIIB and RNA polymerase II-associated factor), Ref2, and Syc1 are associated with the Pta1 subunit of CPF. Systematic tagging and purification of holo-CPF subunits revealed that yeast extracts contain similar amounts of CPF and holo-CPF. By purifying holo-CPF from strains lacking Ref2 or containing truncated subunits, subcomplexes were isolated that revealed additional aspects of the architecture of APT and holo-CPF. Chromatin immunoprecipitation was used to localize Ref2, Ssu72, Pta1, and other APT subunits on small nucleolar RNA (snoRNA) genes and primarily near the polyadenylation signals of the constitutively expressed PYK1 and PMA1 genes. Use of mutant components of APT revealed that Ssu72 is important for preventing readthrough-dependent expression of downstream genes for both snoRNAs and polyadenylated transcripts. Ref2 and Pta1 similarly affect at least one snoRNA transcript.