isolation-salmonella-typhimurium-containing-phagosomes-from

Isolation of <em>Salmonella typhimurium</em>-containing Phagosomes from Macrophages

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. ...

University of Cologne

Virulent mycobacteria cause arrest of phagosome maturation as a part of their survival strategy in hosts. This process is mediated through multiple virulence factors, whose molecular nature remains elusive. Using Mycobacterium marinum as a model, we performed a genome-wide screen to identify mutants whose ability to inhibit phagosome maturation was impaired, and we succeeded in isolating a comprehensive set of mutants that were not able to occupy an early endosome-like phagosomal compartment in mammalian macrophages. Categorizing and ordering the multiple mutations according to their gene families demonstrated that the genes modulating the cell envelope are the principal factors in arresting phagosome maturation. In particular, we identified a novel gene, pmiA, which is capable of influencing the constitution of the cell envelope lipids, thereby leading to the phagosome maturation block. The pmiA mutant was not able to resist phagosome maturation and was severely attenuated in mice. Complementing the mutant with the wild-type gene restored the attenuated virulence to wild-type levels in mice.

A mycobacterial gene involved in synthesis of an outer cell envelope lipid is a key factor in prevention of phagosome maturation.

Bacterial capsular polysaccharides are virulence factors and are considered T cell-independent antigens. However, the capsular polysaccharide Sp1 from Streptococcus pneumoniae serotype 1 has been shown to activate CD4(+) T cells in a major histocompatibility complex (MHC) class II-dependent manner. The mechanism of carbohydrate presentation to CD4(+) T cells is unknown. We show in live murine dendritic cells (DCs) that Sp1 translocates from lysosomal compartments to the plasma membrane in MHCII-positive tubules. Sp1 cell surface presentation results in reduction of self-peptide presentation without alteration of the MHCII self peptide repertoire. In DM-deficient mice, retrograde transport of Sp1/MHCII complexes resulting in T cell-dependent immune responses to the polysaccharide in vitro and in vivo is significantly reduced. The results demonstrate the capacity of a bacterial capsular polysaccharide antigen to use DC tubules as a vehicle for its transport as an MHCII/saccharide complex to the cell surface for the induction of T cell activation. Furthermore, retrograde transport requires the functional role of DM in self peptide-carbohydrate exchange. These observations open new opportunities for the design of vaccines against microbial encapsulated pathogens.

Transport of Streptococcus pneumoniae capsular polysaccharide in MHC Class II tubules.

Mycobacteriophage L5 is a temperate phage with a broad host range among the fast- and slow-growing mycobacteria such as Mycobacterium smegmatis, Mycobacterium tuberculosis, Mycobacterium avium and Mycobacterium ulcerans. L5 switches off host protein synthesis during the early stage of lytic growth, as was previously shown by protein expression profiling. Also, lethal genetic elements have been identified in L5 based on the fact that transformants could not be obtained with these genes. Using an inducible mycobacterial shuttle vector, we have identified three ORFs within an early operon of mycobacteriophage L5 which encode gene products (gp) toxic to the host M. smegmatis when expressed. These ORFs, coding for gp77, gp78 and gp79, presumably function as shut-off genes during early stages of phage replication. There is evidence that cell division is affected by one of the proteins (gp79). The transcription of the cytotoxic polypeptides is directed by a promoter situated in ORF83 and transcription control is achieved through the phage repressor gp71, which is shown by co-expression of this protein. The findings presented here should provide useful tools for the molecular genetics of mycobacteria. Further analysis of these and other mycobacteriophage-derived toxic polypeptides, together with the identification of their cellular targets, might provide a tool for the rapid identification of promising drug targets in emerging and re-emerging mycobacterial pathogens.

Identification of three cytotoxic early proteins of mycobacteriophage L5 leading to growth inhibition in Mycobacterium smegmatis.

Inhibition of phagosome maturation is an important hallmark of mycobacterial pathogenesis. A variety of genomic, transcriptomic and proteomic approaches have been used to pin down the molecule responsible for this pathogenic principle. We in this study characterize a glycolipid of Mycobacterium marinum identified through a screen of mutants disabled in inhibiting phagosome maturation to be phenolphthiocerol diester (phenolic glycolipid, PGL). This molecule is sufficient to impart its ability to inhibit phagosome maturation onto other microbial cells and even inert beads that are used as model pathogens. In addition, it abrogates pro-inflammatory cytokine secretion induced by strong inducers such as heat-killed Mycobacterium bovis bacille Calmette-Guérin. This strong dual agonistic effect of PGL overrides pro-inflammatory and pro-lysosomal delivery impulses set not only by mycobacteria but also by other pathogens and thus provides convincing evidence that this molecule is a vital mycobacterial virulence factor.

Mycobacterial phenolic glycolipid inhibits phagosome maturation and subverts the pro-inflammatory cytokine response.

Ag presentation to CD8(+) T cells commences immediately after infection, which facilitates their rapid expansion and control of pathogen. This paradigm is not followed during infection with virulent Salmonella enterica serovar Typhimurium (ST), an intracellular bacterium that causes mortality in susceptible C57BL/6J mice within 7 days and a chronic infection in resistant mice (129 x 1SvJ). Infection of mice with OVA-expressing ST results in the development of a CD8(+) T cell response that is detectable only after the second week of infection despite the early detectable bacterial burden. The mechanism behind the delayed CD8(+) T cell activation was evaluated, and it was found that dendritic cells/macrophages or mice infected with ST-OVA failed to present Ag to OVA-specific CD8(+) T cells. Lack of early Ag presentation was not rescued when mice or dendritic cells/macrophages were infected with an attenuated aroA mutant of ST or with mutants having defective Salmonella pathogenicity island I/II genes. Although extracellular ST proliferated extensively, the replication of ST was highly muted once inside macrophages. This muted intracellular proliferation of ST resulted in the generation of poor levels of intracellular Ag and peptide-MHC complex on the surface of dendritic cells. Additional experiments revealed that ST did not actively inhibit Ag presentation, rather it inhibited the uptake of another intracellular pathogen, Listeria monocytogenes, thereby causing inhibition of Ag presentation against L. monocytogenes. Taken together, this study reveals a dichotomy in the proliferation of ST and indicates that selectively reduced intracellular proliferation of virulent pathogens may be an important mechanism of immune evasion.

Selectively reduced intracellular proliferation of Salmonella enterica serovar typhimurium within APCs limits antigen presentation and development of a rapid CD8 T cell response.

Food-borne infections caused by Salmonella enterica species are increasing globally, and pregnancy poses a high risk. Pregnant mice rapidly succumb to S. enterica serovar Typhimurium infection. To determine the mechanisms involved, we addressed the role of inflammation and bacterial burden in causing placental and systemic disease. In vitro, choriocarcinoma cells were a highly conducive niche for intracellular S. Typhimurium proliferation. While infection of mice with S. Typhimurium wild-type (WT) and mutant (Delta aroA and Delta invA) strains led to profound pathogen proliferation and massive burden within placental cells, only the virulent WT S. Typhimurium infection evoked total fetal loss and adverse host outcome. This correlated with substantial placental expression of granulocyte colony-stimulating factor (G-CSF), interleukin-6 (IL-6), and tumor necrosis factor alpha (TNF-alpha) and increased serum inflammatory cytokines/chemokines, such as G-CSF, IL-6, CCL1, and KC, evoked by WT S. Typhimurium infection. In contrast, infection with high doses of S. Typhimurium Delta aroA, despite causing massive placental infection, resulted in reduced inflammatory cellular and cytokine response. While S. Typhimurium WT bacteria were dispersed in large numbers across all regions of the placenta, including the deeper labyrinth trophoblast, S. Typhimurium Delta aroA bacteria localized primarily to the decidua. This correlated with the widespread placental necrosis accompanied by neutrophil infiltration evoked by the S. Typhimurium WT bacteria. Thus, the ability of Salmonella to localize to deeper layers of the placenta and the nature of inflammation triggered by the pathogen, rather than bacterial burden, profoundly influenced placental integrity and host survival.

Salmonella enterica serovar Typhimurium-induced placental inflammation and not bacterial burden correlates with pathology and fatal maternal disease.

WhiB-like proteins of actinomycetes are known to co-ordinate iron-sulfur (Fe-S) clusters and are believed to have regulatory functions in many essential bacterial processes. The systematic determination of the genome sequences of mycobacteriophages has revealed the presence of several whiB-like genes in these viruses. Here we focussed on the WhiB-like protein of mycobacteriophage TM4, WhiBTM4. We provide evidence that this viral protein is capable of co-ordinating a Fe-S cluster. The UV-visible absorption spectra obtained from freshly purified and reconstituted WhiBTM4 were consistent with the presence of an oxygen sensitive [2Fe-2S] cluster. Expression of WhiBTM4 in the mycobacterial host led to hindered septation resembling a WhiB2 knockout phenotype whereas basal expression of WhiBTM4 led to superinfection exclusion. The quantification of mRNA-levels during phage infection showed that whiBTM4 is a highly transcribed early phage gene and a dominant negative regulator of WhiB2. Strikingly, both apo-WhiB2 of Mycobacterium tuberculosis and apo-WhiBTM4 were capable of binding to the conserved promoter region upstream of the whiB2 gene indicating that WhiB2 regulates its own synthesis which is inhibited in the presence of WhiBTM4. Thus, we provide substantial evidence supporting the hypothesis of viral and bacterial WhiB proteins being important Fe-S containing transcriptional regulators with DNA-binding capability.

Insights into the function of the WhiB-like protein of mycobacteriophage TM4--a transcriptional inhibitor of WhiB2.

Salmonella enterica serovar Typhimurium (S. Typhimurium) is a virulent pathogen that induces rapid host death. Here we observed that host survival after infection with S. Typhimurium was enhanced in the absence of type I interferon signaling, with improved survival of mice deficient in the receptor for type I interferons (Ifnar1(-/-) mice) that was attributed to macrophages. Although there was no impairment in cytokine expression or inflammasome activation in Ifnar1(-/-) macrophages, they were highly resistant to S. Typhimurium-induced cell death. Specific inhibition of the kinase RIP1 or knockdown of the gene encoding the kinase RIP3 prevented the death of wild-type macrophages, which indicated that necroptosis was a mechanism of cell death. Finally, RIP3-deficient macrophages, which cannot undergo necroptosis, had similarly less death and enhanced control of S. Typhimurium in vivo. Thus, we propose that S. Typhimurium induces the production of type I interferon, which drives necroptosis of macrophages and allows them to evade the immune response.

Type I interferon induces necroptosis in macrophages during infection with Salmonella enterica serovar Typhimurium.

A pregnant woman with chronic meningococcaemia from Neisseria meningitidis with lpxL1-mutations.

The influence of sex hormones is recognized to account for the susceptibility and distinct outcomes of diverse infectious diseases.

Sex differences in immune responses to infectious diseases.

During intracellular infections, autophagy significantly contributes to the elimination of pathogens, regulation of pro-inflammatory signaling, secretion of immune mediators and in coordinating the adaptive immune system. Intracellular pathogens such as S. Typhimurium have evolved mechanisms to circumvent autophagy. However, the regulatory mechanisms targeted by S. Typhimurium to modulate autophagy have not been fully resolved. Here we report that cytosolic energy loss during S. Typhimurium infection triggers transient activation of AMPK, an important checkpoint of mTOR activity and autophagy. The activation of AMPK is regulated by LKB1 in a cytosolic complex containing Sirt1 and LKB1, where Sirt1 is required for deacetylation and subsequent activation of LKB1. S. Typhimurium infection targets Sirt1, LKB1 and AMPK to lysosomes for rapid degradation resulting in the disruption of the AMPK-mediated regulation of mTOR and autophagy. The degradation of cytosolic Sirt1/LKB1/AMPK complex was not observed with two mutant strains of S. Typhimurium, ΔssrB and ΔssaV, both compromising the pathogenicity island 2 (SPI2). The results highlight virulence factor-dependent degradation of host cell proteins as a previously unrecognized strategy of S. Typhimurium to evade autophagy.

Article	Year
A mycobacterial gene involved in synthesis of an outer cell envelope lipid is a key factor in prevention of phagosome maturation. Infection and immunity\| PubMed ID: 17088345	2007
Transport of Streptococcus pneumoniae capsular polysaccharide in MHC Class II tubules. PLoS pathogens\| PubMed ID: 17367207	2007
Identification of three cytotoxic early proteins of mycobacteriophage L5 leading to growth inhibition in Mycobacterium smegmatis. Microbiology (Reading, England)\| PubMed ID: 18667563	2008
Mycobacterial phenolic glycolipid inhibits phagosome maturation and subverts the pro-inflammatory cytokine response. Traffic (Copenhagen, Denmark)\| PubMed ID: 18764820	2008
Selectively reduced intracellular proliferation of Salmonella enterica serovar typhimurium within APCs limits antigen presentation and development of a rapid CD8 T cell response. Journal of immunology (Baltimore, Md. : 1950)\| PubMed ID: 19692639	2009
Salmonella enterica serovar Typhimurium-induced placental inflammation and not bacterial burden correlates with pathology and fatal maternal disease. Infection and immunity\| PubMed ID: 20194592	2010
Insights into the function of the WhiB-like protein of mycobacteriophage TM4--a transcriptional inhibitor of WhiB2. Molecular microbiology\| PubMed ID: 20545868	2010
Type I interferon induces necroptosis in macrophages during infection with Salmonella enterica serovar Typhimurium. Nature immunology\| PubMed ID: 22922364	2012
A pregnant woman with chronic meningococcaemia from Neisseria meningitidis with lpxL1-mutations. Lancet (London, England)\| PubMed ID: 25457917	2014
Sex differences in immune responses to infectious diseases. Infection\| PubMed ID: 25956991	2015
Salmonella Typhimurium disrupts Sirt1/AMPK checkpoint control of mTOR to impair autophagy. PLoS pathogens\| PubMed ID: 28192515	2017

Nirmal Robinson

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