Brucella Induces an Unfolded Protein Response via TcpB That Supports Intracellular Replication in Macrophages

Brucella melitensis is a facultative intracellular bacterium that causes brucellosis, the most prevalent zoonosis worldwide. The Brucella intracellular replicative niche in macrophages and dendritic cells thwarts immune surveillance and complicates both therapy and vaccine development. Currently, host-pathogen interactions supporting Brucella replication are poorly understood. Brucella fuses with the endoplasmic reticulum (ER) to replicate, resulting in dramatic restructuring of the ER. This ER disruption raises the possibility that Brucella provokes an ER stress response called the Unfolded Protein Response (UPR). In this study, B. melitensis infection up regulated expression of the UPR target genes BiP, CHOP, and ERdj4, and induced XBP1 mRNA splicing in murine macrophages. These data implicate activation of all 3 major signaling pathways of the UPR. Consistent with previous reports, XBP1 mRNA splicing was largely MyD88-dependent. However, up regulation of CHOP, and ERdj4 was completely MyD88 independent. Heat killed Brucella stimulated significantly less BiP, CHOP, and ERdj4 expression, but induced XBP1 splicing. Although a Brucella VirB mutant showed relatively intact UPR induction, a TcpB mutant had significantly compromised BiP, CHOP and ERdj4 expression. Purified TcpB, a protein recently identified to modulate microtubules in a manner similar to paclitaxel, also induced UPR target gene expression and resulted in dramatic restructuring of the ER. In contrast, infection with the TcpB mutant resulted in much less ER structural disruption. Finally, tauroursodeoxycholic acid, a pharmacologic chaperone that ameliorates the UPR, significantly impaired Brucella replication in macrophages. Together, these results suggest Brucella induces a UPR, via TcpB and potentially other factors, that enables its intracellular replication. Thus, the UPR may provide a novel therapeutic target for the treatment of brucellosis. These results also have implications for other intracellular bacteria that rely on host physiologic stress responses for replication.     

The role of endoplasmic reticulum‐related BiP/GRP78 in interferon gamma‐induced persistent Chlamydia pneumoniae infection

Direct interaction of Chlamydiae with the endoplasmic reticulum (ER) is essential in intracellular productive infection. However, little is known about the interplay between Chlamydiae and the ER under cellular stress conditions that are observed in interferon gamma (IFN‐γ) induced chlamydial persistent infection. ER stress responses are centrally regulated by the unfolded protein response (UPR) under the control of the ER chaperone BiP/GRP78 to maintain cellular homeostasis. In this study, we could show that the ER directly contacted with productive and IFN‐γ‐induced persistent inclusions of Chlamydia pneumoniae (Cpn). BiP/GRP78 induction was observed in the early phase but not in the late phase of IFN‐γ‐induced persistent infection. Enhanced BiP/GRP78 expression in the early phase of IFN‐γ‐induced persistent Cpn infection was accompanied by phosphorylation of the eukaryotic initiation factor‐2α (eIF2α) and down‐regulation of the vesicle‐associated membrane protein‐associated protein B. Loss of BiP/GRP78 function resulted in enhanced phosphorylation of eIF2α and increased host cell apoptosis. In contrast, enhanced BiP/GRP78 expression in IFN‐γ‐induced persistent Cpn infection attenuated phosphorylation of eIF2α upon an exogenous ER stress inducer. In conclusion, ER‐related BiP/GRP78 plays a key role to restore cells from stress conditions that are observed in the early phase of IFN‐γ‐induced persistent infection.     

Repression of bacterial lipoprotein production by Francisella novicida facilitates evasion of innate immune recognition

Innate recognition systems, including the Toll‐like receptors (TLRs), play a critical role in activating host defences and proinflammatory pathways in response to infection. Pathogens have developed strategies to subvert TLRs in order to survive and replicate within the host. The model intracellular pathogen, Francisella novicida, modulates host defences to promote survival and replication in macrophages. TLR2, which recognizes bacterial lipoproteins (BLPs), is critical for activating host defences and proinflammatory cytokine production in response to Francisella infection. Here we show that the F. novicida protein FTN_0757 acts to repress BLP production, dampening TLR2 activation. The ΔFTN_0757 mutant strain induced robust TLR2‐dependent cytokine production in macrophages compared with wild‐type bacteria, and produced increased amounts of BLPs. The deletion of one BLP (FTN_1103) from ΔFTN_0757 decreased the total BLP concentration to near wild‐type level sand correlated with a decrease in the inductionof TLR2 signalling. The overproduction of BLPs also contributed to the in vivo attenuation of the ΔFTN_0757 mutant, which was significantly rescued when FTN_1103 was deleted. Taken together, these data reveal a novel mechanism of immune evasion by the downregulation of BLP expression to subvert TLR2 activation, which is likely used by numerous other intracellular bacterial pathogens.     

Francisella tularensis regulates the expression of the amino acid transporter SLC1A5 in infected THP‐1 human monocytes

Francisella tularensis, a Gram‐negative bacterium that causes the disease tularemia in a large number of animal species, is thought to reside preferentially within macrophages in vivo. F. tularensis has developed mechanisms to rapidly escape from the phagosome into the cytoplasm of infected cells, a habitat with a rich supply of nutrients, ideal for multiplication. SLC1A5 is a neutral amino acid transporter expressed by human cells, which serves, along with SLC7A5 to equilibrate cytoplasmic amino acid pools. We herein analysed whether SLC1A5 was involved in F. tularensis intracellular multiplication. We demonstrate that expression of SLC1A5 is specifically upregulated by F. tularensis in infected THP‐1 human monocytes. Furthermore, we show that SLC1A5 downregulation decreases intracellular bacterial multiplication, supporting the involvement of SLC1A5 in F. tularensis infection. Notably, after entry of F. tularensis into cells and during the whole infection, the highly glycosylated form of SLC1A5 was deglycosylated only by bacteria capable of cytosolic multiplication. These data suggest that intracellular replication of F. tularensis depends on the function of host cell SLC1A5. Our results are the first, which show that Francisella intracellular multiplication in human monocyte cytoplasm is associated with a post‐translational modification of a eukaryotic amino acid transporter.     

Cell biology and molecular ecology of Francisella tularensis

Francisella tularensis is a highly infectious intracellular bacterium that causes the fulminating disease tularemia, which can be transmitted between mammals by arthorpod vectors. Genomic studies have shown that the F. tularensis has been undergoing genomic decay with the most virulent strains having the lowest number of functional genes. Entry of F. tularensis into macrophages is mediated by looping phagocytosis and is associated with signalling through Syk tyrosine kinase. Within macrophages and arthropod‐derived cells, the Francisella‐containing phagosome matures transiently into an acidified late endosome‐like phagosome with limited fusion to lysosomes followed by rapid bacterial escape into the cytosol within 30–60 min, and bacterial proliferation within the cytosol. The Francisella pathogenicity island, which potentially encodes a putative type VI secretion system, is essential for phagosome biogenesis and bacterial escape into the cytosol within macrophages and arthropod‐derived cells. Initial sensing of F. tularensis in the cytosol triggers IRF‐3‐dependent IFN‐β secretion, type I IFNR‐dependent signalling, activation of the inflammasome mediated by caspase‐1, and a pro‐inflammatory response, which is suppressed by triggering of SHIP. The past few years have witnessed a quantum leap in our understanding of various aspects of this organism and this review will discuss these remarkable advances.     

Molecular complexity orchestrates modulation of phagosome biogenesis and escape to the cytosol of macrophages by Francisella tularensis

Upon entry of Francisella tularensis to macrophages, the Francisella‐containing phagosome (FCP) is trafficked into an acidified late endosome‐like phagosome with limited fusion to the lysosomes followed by rapid escape into the cytosol where the organism replicates. Although the Francisella Pathogenicity Island (FPI), which encodes a type VI‐like secretion apparatus, is required for modulation of phagosome biogenesis and escape into the cytosol, the mechanisms involved are not known. To decipher the molecular bases of modulation of biogenesis of the FCP and bacterial escape into the macrophage cytosol, we have screened a comprehensive mutant library of F. tularensis ssp. novicida for their defect in proliferation within human macrophages, followed by characterization of modulation of phagosome biogenesis and bacterial escape into the cytosol. Our data show that at least 202 genes are required for intracellular proliferation within macrophages. Among the 125 most defective mutants in intracellular proliferation, we show that the FCP of at least 91 mutants colocalize persistently with the late endosomal/lysosomal marker LAMP‐1 and fail to escape into the cytosol, as determined by fluorescence‐based phagosome integrity assays and transmission electron microscopy. At least 34 genes are required for proliferation within the cytosol but do not play a detectable role in modulation of phagosome biogenesis and bacterial escape into the cytosol. Our data indicate a tremendous adaptation and metabolic reprogramming by F. tularensis to adjust to the micro‐environmental and nutritional cues within the FCP, and these adjustments play essential roles in modulation of phagosome biogenesis and escape into the cytosol of macrophages as well as proliferation in the cytosol. The plethora of the networks of genes that orchestrate F. tularensis‐mediated modulation of phagosome biogenesis, phagosomal escape and bacterial proliferation within the cytosol is novel, complex and involves an unusually large portion of the genome of an intracellular pathogen.     

Microbial community structure reveals instability of nutritional symbiosis during the evolutionary radiation of Amblyomma ticks

Mutualistic interactions with microbes have facilitated the adaptation of major eukaryotic lineages to restricted diet niches. Hence, ticks with their strictly blood‐feeding lifestyle are associated with intracellular bacterial symbionts through an essential B vitamin supplementation. In this study, examination of bacterial diversity in 25 tick species of the genus Amblyomma showed that three intracellular bacteria, Coxiella‐like endosymbionts (LE), Francisella‐LE and Rickettsia, are remarkably common. No other bacterium is as uniformly present in Amblyomma ticks. Almost all Amblyomma species were found to harbour a nutritive obligate symbiont, Coxiella‐LE or Francisella‐LE, that is able to synthesize B vitamins. However, despite the co‐evolved and obligate nature of these mutualistic interactions, the structure of microbiomes does not mirror the Amblyomma phylogeny, with a clear exclusion pattern between Coxiella‐LE and Francisella‐LE across tick species. Coxiella‐LE, but not Francisella‐LE, form evolutionarily stable associations with ticks, commonly leading to co‐cladogenesis. We further found evidence for symbiont replacements during the radiation of Amblyomma, with recent, and probably ongoing, invasions by Francisella‐LE and subsequent replacements of ancestral Coxiella‐LE through transient co‐infections. Nutritional symbiosis in Amblyomma ticks is thus not a stable evolutionary state, but instead arises from conflicting origins between unrelated but competing symbionts with similar metabolic capabilities.     

Molecular bases of proliferation of Francisella tularensis in arthropod vectors

Arthropod vectors are important vehicles for transmission of Francisella tularensis between mammals, but very little is known about the F. tularensis–arthropod vector interaction. Drosophila melanogaster has been recently developed as an arthropod vector model for F. tularensis. We have shown that intracellular trafficking of F. tularensis within human monocytes‐derived macrophages and D. melanogaster‐derived S2 cells is very similar. Within both evolutionarily distant host cells, the Francisella‐containing phagosome matures to a late endosome‐like phagosome with limited fusion to lysosomes followed by rapid bacterial escape into the cytosol where the bacterial proliferate. To decipher the molecular bases of intracellular proliferation of F. tularensis within arthropod‐derived cells, we screened a comprehensive library of mutants of F. tularensis ssp. novicida for their defect in intracellular proliferation within D. melanogaster‐derived S2 cells. Our data show that 394 genes, representing 22% of the genome, are required for intracellular proliferation within D. melanogaster‐derived S2 cells, including many of the Francisella Pathogenicity Island (FPI) genes that are also required for proliferation within mammalian macrophages. Functional gene classes that exhibit growth defect include metabolic (25%), FPI (2%), type IV pili (1%), transport (16%) and DNA modification (5%). Among 168 most defective mutants in intracellular proliferation in S2 cells, 80 are defective in lethality and proliferation within adult D. melanogaster. The observation that only 135 of the 394 mutants that are defective in S2 cells are also defective in human macrophages indicates that F. tularensis utilize common as well as distinct mechanisms to proliferate within mammalian and arthropod cells. Our studies will facilitate deciphering the molecular aspects of F. tularensis–arthropod vector interaction and its patho‐adaptation to infect mammals.     

Azithromycin effectiveness against intracellular infections of <Emphasis Type="Italic">Francisella</Emphasis>

Background  

Macrolide antibiotics are commonly administered for bacterial respiratory illnesses. Azithromycin (Az) is especially noted for extremely high intracellular concentrations achieved within macrophages which is far greater than the serum concentration. Clinical strains of Type B Francisella (F.) tularensis have been reported to be resistant to Az, however our laboratory Francisella strains were found to be sensitive. We hypothesized that different strains/species of Francisella (including Type A) may have different susceptibilities to Az, a widely used and well-tolerated antibiotic.     

Triggering Ras signalling by intracellular Francisella tularensis through recruitment of PKCα and βI to the SOS2/GrB2 complex is essential for bacterial proliferation in the cytosol

Intracellular proliferation of Francisella tularensis is essential for manifestation of the fatal disease tularaemia, and is classified as a category A bioterrorism agent. The F. tularensis‐containing phagosome (FCP) matures into a late endosome‐like phagosome with limited fusion to lysosomes, followed by rapid bacterial escape into the cytosol. The Francisella pathogenicity island (FPI) encodes a type VI‐like secretion system, and the FPI‐encoded IglC is essential for evasion of lysosomal fusion and phagosomal escape. Many host signalling events are likely to be modulated by F. tularensis to render the cell permissive for intracellular proliferation but they are not fully understood. Here we show that within 15 min of infection, intracellular F. tularensis ssp. novicida triggers IglC‐dependent temporal activation of Ras, but attached extracellular bacteria fail to trigger Ras activation, which has never been shown for other intracellular pathogens. Intracellular F. tularensis ssp. novicida triggers activation of Ras through recruitment of PKCα and PKCβI to the SOS2/GrB2 complex. Silencing of SOS2, GrB2 and PKCα and PKCβI by RNAi has no effect on evasion of lysosomal fusion and bacterial escape into the cytosol but renders the cytosol non‐permissive for replication of F. tularensis ssp. novicida. Since Ras activation promotes cell survival, we show that silencing of SOS2, GrB2 and PKCα and βI is associated with rapid early activation of caspase‐3 within 8 h post infection. However, silencing of SOS2, GrB2 and PKCα and βI does not affect phosphorylation of Akt or Erk, indicating that activation of the PI3K/Akt and the Erk signalling cascade are independent of the F. tularensis‐triggered Ras activation. We conclude that intracellular F. tularensis ssp. novicida triggers temporal and early activation of Ras through the SOS2/GrB2/PKCα/PKCβI quaternary complex. Temporal and rapid trigger of Ras signalling by intracellular F. tularensis is essential for intracellular bacterial proliferation within the cytosol, and this is associated with downregulation of early caspase‐3 activation.     

TGBp3 triggers the unfolded protein response and SKP1‐dependent programmed cell death

The Potato virus X (PVX) triple gene block protein 3 (TGBp3), an 8‐kDa membrane binding protein, aids virus movement and induces the unfolded protein response (UPR) during PVX infection. TGBp3 was expressed from the Tobacco mosaic virus (TMV) genome (TMV‐p3), and we noted the up‐regulation of SKP1 and several endoplasmic reticulum (ER)‐resident chaperones, including the ER luminal binding protein (BiP), protein disulphide isomerase (PDI), calreticulin (CRT) and calmodulin (CAM). Local lesions were seen on leaves inoculated with TMV‐p3, but not TMV or PVX. Such lesions were the result of TGBp3‐elicited programmed cell death (PCD), as shown by an increase in reactive oxygen species, DNA fragmentation and induction of SKP1 expression. UPR‐related gene expression occurred within 8 h of TMV‐p3 inoculation and declined before the onset of PCD. TGBp3‐mediated cell death was suppressed in plants that overexpressed BiP, indicating that UPR induction by TGBp3 is a pro‐survival mechanism. Anti‐apoptotic genes Bcl‐xl, CED‐9 and Op‐IAP were expressed in transgenic plants and suppressed N gene‐mediated resistance to TMV, but failed to alleviate TGBp3‐induced PCD. However, TGBp3‐mediated cell death was reduced in SKP1‐silenced Nicotiana benthamiana plants. The combined data suggest that TGBp3 triggers the UPR and elicits PCD in plants.     

Leishmania donovani infection differentially regulates small G‐proteins

Leishmania is a protozoan parasite that resides and replicates in macrophages and causes leishmaniasis. The parasite alters the signaling cascade in host macrophages and evades the host machinery. Small G‐proteins are GTPases, grouped in 5 different families that play a crucial role in the regulation of cell proliferation, cell survival, apoptosis, intracellular trafficking, and transport. In particular, the Ras family of small G‐proteins has been identified to play a significant role in the cellular functions mentioned before. Here, we studied the differential expression of the most important small G‐proteins during Leishmania infection. We found major changes in the expression of different isoforms of Ras, mainly in N‐Ras. We observed that Leishmania donovani infection led to enhanced N‐Ras expression, whereas it inhibited K‐Ras and H‐Ras expression. Furthermore, an active N‐Ras pull‐down assay showed enhanced N‐Ras activity. L donovani infection also increased extracellular signal–regulated kinase 1/2 phosphorylation and simultaneously decreased p38 phosphorylation. In contrast, pharmacological inhibition of Ras led to reduction in the phosphorylation of extracellular signal–regulated kinase 1/2 and enhanced the phosphorylation of p38 in Leishmania‐infected cells, which could lead to increased interleukin‐12 expression and decreased interleukin‐10 expression. Indeed, farnesylthiosalicyclic acid (a Ras inhibitor), when used at the effective level in L donovani–infected macrophages, reduced amastigotes in the host macrophages. Thus, upregulated N‐Ras expression during L donovani infection could be a novel immune evasion strategy of Leishmania and would be a potential target for antileishmanial immunotherapy.     

Precision mapping of the human O‐GalNAc glycoproteome through SimpleCell technology

Glycosylation is the most abundant and diverse posttranslational modification of proteins. While several types of glycosylation can be predicted by the protein sequence context, and substantial knowledge of these glycoproteomes is available, our knowledge of the GalNAc‐type O‐glycosylation is highly limited. This type of glycosylation is unique in being regulated by 20 polypeptide GalNAc‐transferases attaching the initiating GalNAc monosaccharides to Ser and Thr (and likely some Tyr) residues. We have developed a genetic engineering approach using human cell lines to simplify O‐glycosylation (SimpleCells) that enables proteome‐wide discovery of O‐glycan sites using ‘bottom‐up’ ETD‐based mass spectrometric analysis. We implemented this on 12 human cell lines from different organs, and present a first map of the human O‐glycoproteome with almost 3000 glycosites in over 600 O‐glycoproteins as well as an improved NetOGlyc4.0 model for prediction of O‐glycosylation. The finding of unique subsets of O‐glycoproteins in each cell line provides evidence that the O‐glycoproteome is differentially regulated and dynamic. The greatly expanded view of the O‐glycoproteome should facilitate the exploration of how site‐specific O‐glycosylation regulates protein function.     

Long‐term live‐cell imaging reveals new roles for Salmonella effector proteins SseG and SteA

Salmonella Typhimurium is an intracellular bacterial pathogen that infects both epithelial cells and macrophages. Salmonella effector proteins, which are translocated into the host cell and manipulate host cell components, control the ability to replicate and/or survive in host cells. Due to the complexity and heterogeneity of Salmonella infections, there is growing recognition of the need for single‐cell and live‐cell imaging approaches to identify and characterize the diversity of cellular phenotypes and how they evolve over time. Here, we establish a pipeline for long‐term (17 h) live‐cell imaging of infected cells and subsequent image analysis methods. We apply this pipeline to track bacterial replication within the Salmonella‐containing vacuole in epithelial cells, quantify vacuolar replication versus survival in macrophages and investigate the role of individual effector proteins in mediating these parameters. This approach revealed that dispersed bacteria can coalesce at later stages of infection, that the effector protein SseG influences the propensity for cytosolic hyper‐replication in epithelial cells, and that while SteA only has a subtle effect on vacuolar replication in epithelial cells, it has a profound impact on infection parameters in immunocompetent macrophages, suggesting differential roles for effector proteins in different infection models.     

Rice stripe virus activates the bZIP17/28 branch of the unfolded protein response signalling pathway to promote viral infection

The unfolded protein response (UPR) plays important roles in plant virus infection. Our previous study has proved that rice stripe virus (RSV) infection elicits host UPR. However, the mechanism on how the UPR is triggered upon RSV infection remains obscure. Here, we show that the bZIP17/28 branch of the UPR signalling pathway is activated upon RSV infection in Nicotiana benthamiana. We found that membrane-associated proteins NSvc2 and NSvc4 encoded by RSV are responsible for the activation of the bZIP17/28 branch. Ectopic expression of NSvc2 or NSvc4 in plant leaves induced the proteolytic processing of NbbZIP17/28 and up-regulated the expression of UPR-related genes. Silencing NbbZIP17/28 significantly inhibited RSV infection. We show that RSV can specifically elicit the UPR through the bZIP17/28 branch, thus promoting virus infection of N. benthamiana plants.     

Analysis of unfolded protein response during single-chain antibody expression in Saccaromyces cerevisiae reveals different roles for BiP and PDI in folding

The production of recombinant proteins is a critical technology for biotechnology and biomedical research. Heterologous expression of secreted proteins can saturate the cell's capacity to properly fold protein, initiating the unfolded protein response (UPR), and resulting in a loss of protein expression. The overexpression of chaperone binding protein (BiP) and disulfide bond isomerase (PDI) in Saccaromyces cerevisiae can effectively increase protein production levels of single-chain antibody (scFv) 4-4-20. These studies show that overexpression of BiP did not reduce the UPR activated by heterologous protein expression; however, overexpression of PDI or co-overexpression of BiP and PDI could reduce the UPR. We observed that co-overexpression of BiP and PDI led to the greatest secretion of scFv from the cell, but BiP and PDI appear to interact with the newly synthesized scFv at different stages in the folding process, as determined by pulse-chase analysis. We propose that BiP acts primarily to facilitate translocation and retain unfolded or partially folded scFv, and PDI actively folds the scFv through its functions as a catalyst, and/or an isomerase, of disulfide bonds. Free BiP is released when scFv is folded, stabilizing Ire1p, and leading to the reduced UPR.