Intracellular proliferation of Anaplasma phagocytophilum is promoted via modulation of endoplasmic reticulum stress signaling in host cells.

Anaplasma phagocytophilum, an obligate intracellular bacterium that propagates within host granulocytes, is considered to modify the host intracellular environment for pathogenesis. However, the mechanism(s) underlying such host modifications remain unclear. Here, we aimed to investigate the relation between A. phagocytophilum and endoplasmic reticulum (ER) stress in THP-1 cells. A. phagocytophilum activated the three ER stress sensors: inositol-requiring enzyme-1 (IRE1), protein kinase RNA-like endoplasmic reticulum kinase (PERK), and activating transcription factor-6 (ATF6). IRE1 activation occurred immediately after host cell invasion by A. phagocytophilum; however, the activated IRE1-induced splicing of X-box-binding protein 1 was not promoted during A. phagocytophilum infection. This suppression was sustained even after the doxycycline-mediated elimination of intracellular A. phagocytophilum. IRE1 knockdown accelerated A. phagocytophilum-induced apoptosis and decreased intracellular A. phagocytophilum. These data suggest that A. phagocytophilum utilizes IRE1 activation to promote its own intracellular proliferation. Moreover, PERK and ATF6 partially mediated A. phagocytophilum-induced apoptosis by promoting the expression of CCAAT/enhancer-binding protein homologous protein, which induces the transcription of several proapoptotic genes. Thus, A. phagocytophilum possibly manipulates the host ER stress signals to facilitate intracellular proliferation and infection of surrounding cells before/after host cell apoptosis.

Devitalization of the immune mapped protein 1 undermines the intracellular proliferation of Toxoplasma gondii.

The intracellular protozoan Toxoplasma gondii infects approximately one-third of the world's population as well as various animals, causing toxoplasmosis. However, there remains a need to define the functions of newly identified genes of T. gondii. In the present study, a novel molecule, immune mapped protein 1 of T. gondii (TgIMP1), was devitalized by CRISPR/Cas9 system to investigate the phenotypic changes of the parasite. We found that the virulence of ΔTgIMP1 knockout strain was reduced in comparison with wild-type GT1 tachyzoites, showing a statistically decreased plaque in HFF cells and a significantly prolonged survival period of mice (P < 0.05). Moreover, the data of phenotype analyses in vitro showed a different level of the intracellular proliferation and the subsequent egress between ΔTgIMP1 and wild-type GT1 strain (P < 0.05); while no statistically significant difference was detected during the process of attachment or invasion. These results suggested that TgIMP1 is closely associated with the intracellular proliferation of this parasite.

TgERK7 is involved in the intracellular proliferation of Toxoplasma gondii.

Toxoplasma gondii uses a unique mechanism to fulfill its asexual life cycles by which the parasite can infect all the warm-blooded animals including humans. Mitogen-activated protein kinase (MAPK) or extracellular signal-regulated kinase (ERK) pathway widely existed in eukaryotic cells mediates the conversion of environmental stimuli to intracellular events such as proliferation and differentiation. Their counterparts have been identified in Apicomplexan parasites such as ERK7 in T. gondii. To confirm whether the unique mechanism of T. gondii is relevant to MAPK/ERK member, we created a mutant (ΔTgERK7) in GT1 tachyzoites using double homologous recombination method. Our results of virulence evaluation showed 100 % survival of all the ΔTgERK7-infected mice until 35 days post-challenge compared to no survival in wild-type GT1-infected group (10.6 ± 0.34 days). Furthermore, lower parasite loads were detected in the peritoneal fluid of ΔTgERK7-infected mice (P < 0.05). To ensure whether or not ERK7 gene knockout leads to the growth deficiency of T. gondii, the intracellular proliferation of ΔTgERK7 was also examined in vitro. Our data indicated that the proliferation of ΔTgERK7 parasites was significantly prolonged in comparison with wild-type GT1 tachyzoites (P < 0.05). Therefore, we concluded that TgERK7 is important for the intracellular proliferation of T. gondii, which further emphasized that MAPK/ERK derived from T. gondii participates in the regulation of the asexual life cycles to ensure the survival and reinfections of this parasite.

The DNA adenine methylase of Salmonella Enteritidis promotes their intracellular replication by inhibiting arachidonic acid metabolism pathway in macrophages.

Macrophages can participate in immune responses by altering their metabolism, and play important roles in controlling bacterial infections. However, Salmonella Enteritidis can survive and proliferate in macrophages. After the deletion of DNA adenine methylase (Dam), the proliferation of Salmonella Enteritidis in macrophages decreased, the molecular mechanism is still unclear. After infecting macrophages with Salmonella Enteritidis wild type and dam gene deletion strains, intracellular metabolites were extracted and detected by non-targeted metabolomics and fatty acid targeted metabolomics. We found Dam had significant effects on arachidonic acid and related metabolic pathways in macrophages. The dam gene can promote the proliferation of Salmonella Enteritidis in macrophages by inhibiting the metabolic pathway of cytosolic phospholipase A2-mediated arachidonic acid production and conversion to prostaglandin E2 in macrophages, reducing the secretion of the pro-inflammatory factors IL-1ß and IL-6. In addition, inhibition of arachidonic acid-related pathways in macrophages by Arachidonyl trifluoromethyl ketone could restore the proliferation of dam gene deletion strains in macrophages. This study explored the role of Dam in the process of Salmonella Enteritidis invading host cells from the perspective of host cell metabolism, and provides new insights into the immune escape mechanism of Salmonella Enteritidis.

Listeria InlB Expedites Vacuole Escape and Intracellular Proliferation by Promoting Rab7 Recruitment via Vps34.

Rapid phagosomal escape mediated by listeriolysin O (LLO) is a prerequisite for Listeria monocytogenes intracellular replication and pathogenesis. Escape takes place within minutes after internalization from vacuoles that are negative to the early endosomal Rab5 GTPase and positive to the late endosomal Rab7. Using mutant analysis, we found that the listerial invasin InlB was required for optimal intracellular proliferation of L. monocytogenes. Starting from this observation, we determined in HeLa cells that InlB promotes early phagosomal escape and efficient Rab7 acquisition by the Listeria-containing vacuole (LCV). Recruitment of the class III phosphoinositide 3-kinase (PI3K) Vps34 to the LCV and accumulation of its lipid product, phosphatidylinositol 3-phosphate (PI3P), two key endosomal maturation mediators, were also dependent on InlB. Small interfering RNA (siRNA) knockdown experiments showed that Vps34 was required for Rab7 recruitment and early (LLO-mediated) escape and supported InlB-dependent intracellular proliferation. Together, our data indicate that InlB accelerates LCV conversion into an escape-favorable Rab7 late phagosome via subversion of class III PI3K/Vps34 signaling. Our findings uncover a new function for the InlB invasin in Listeria pathogenesis as an intracellular proliferation-promoting virulence factor. IMPORTANCE Avoidance of lysosomal killing by manipulation of the endosomal compartment is a virulence mechanism assumed to be largely restricted to intravacuolar intracellular pathogens. Our findings are important because they show that cytosolic pathogens like L. monocytogenes, which rapidly escape the phagosome after internalization, can also extensively subvert endocytic trafficking as part of their survival strategy. They also clarify that, instead of delaying phagosome maturation (to allow time for LLO-dependent disruption, as currently thought), via InlB L. monocytogenes appears to facilitate the rapid conversion of the phagocytic vacuole into an escape-conducive late phagosome. Our data highlight the multifunctionality of bacterial virulence factors. At the cell surface, the InlB invasin induces receptor-mediated phagocytosis via class I PI3K activation, whereas after internalization it exploits class III PI3K (Vsp34) to promote intracellular survival. Systematically elucidating the mechanisms by which Listeria interferes with PI3K signaling all along the endocytic pathway may lead to novel anti-infective therapies.

Mechanisms of Pulmonary Escape and Dissemination by Cryptococcus neoformans.

Cryptococcus neoformans is a common environmental saprophyte and human fungal pathogen that primarily causes disease in immunocompromised individuals. Similar to many environmentally acquired human fungal pathogens, C. neoformans initiates infection in the lungs. However, the main driver of mortality is invasive cryptococcosis leading to fungal meningitis. After C. neoformans gains a foothold in the lungs, a critical early step in invasion is transversal of the respiratory epithelium. In this review, we summarize current knowledge relating to pulmonary escape. We focus on fungal factors that allow C. neoformans to disseminate from the lungs via intracellular and extracellular routes.

Controlled Activity of the Salmonella Invasion-Associated Injectisome Reveals Its Intracellular Role in the Cytosolic Population.

The Salmonella invasion-associated type III secretion system (T3SS1) is an essential virulence factor required for entry into nonphagocytic cells and consequent uptake into a Salmonella-containing vacuole (SCV). While Salmonella is typically regarded as a vacuolar pathogen, a subset of bacteria escape from the SCV in epithelial cells and eventually hyperreplicate in the cytosol. T3SS1 is downregulated following bacterial entry into mammalian cells, but cytosolic Salmonella cells are T3SS1 induced, suggesting prolonged or resurgent activity of T3SS1 in this population. In order to investigate the postinternalization contributions of T3SS1 to the Salmonella infectious cycle in epithelial cells, we bypassed its requirement for bacterial entry by tagging the T3SS1-energizing ATPase InvC at the C terminus with peptides that are recognized by bacterial tail-specific proteases. This caused a dramatic increase in InvC turnover which rendered even assembled injectisomes inactive. Bacterial strains conditionally expressing these unstable InvC variants were proficient for invasion but underwent rapid and sustained intracellular inactivation of T3SS1 activity when InvC expression ceased. This allowed us to directly implicate T3SS1 activity in cytosolic colonization and bacterial egress. We subsequently identified two T3SS1-delivered effectors, SopB and SipA, that are required for efficient colonization of the epithelial cell cytosol. Overall, our findings support a multifaceted, postinvasion role for T3SS1 and its effectors in defining the cytosolic population of intracellular SalmonellaIMPORTANCE A needle-like apparatus, the type III secretion system (T3SS) injectisome, is absolutely required for Salmonella enterica to enter epithelial cells; this requirement has hampered the analysis of its postentry contributions. To identify T3SS1-dependent intracellular activities, in this study we overcame this limitation by developing a conditional inactivation in the T3SS whereby T3SS activity is chemically induced during culture in liquid broth, permitting bacterial entry into epithelial cells, but is quickly and perpetually inactivated in the absence of inducer. In this sense, the mutant acts like wild-type bacteria when extracellular and as a T3SS mutant once it enters a host cell. This "conditional" mutant allowed us to directly link activity of this T3SS with nascent vacuole lysis, cytosolic proliferation, and cellular egress, demonstrating that the invasion-associated T3SS also contributes to essential intracellular stages of the S. enterica infectious cycle.

Microevolutionary traits and comparative population genomics of the emerging pathogenic fungus Cryptococcus gattii.

Emerging fungal pathogens cause an expanding burden of disease across the animal kingdom, including a rise in morbidity and mortality in humans. Yet, we currently have only a limited repertoire of available therapeutic interventions. A greater understanding of the mechanisms of fungal virulence and of the emergence of hypervirulence within species is therefore needed for new treatments and mitigation efforts. For example, over the past decade, an unusual lineage of Cryptococcus gattii, which was first detected on Vancouver Island, has spread to the Canadian mainland and the Pacific Northwest infecting otherwise healthy individuals. The molecular changes that led to the development of this hypervirulent cryptococcal lineage remain unclear. To explore this, we traced the history of similar microevolutionary events that can lead to changes in host range and pathogenicity. Here, we detail fine-resolution mapping of genetic differences between two highly related Cryptococcus gattii VGIIc isolates that differ in their virulence traits (phagocytosis, vomocytosis, macrophage death, mitochondrial tubularization and intracellular proliferation). We identified a small number of single site variants within coding regions that potentially contribute to variations in virulence. We then extended our methods across multiple lineages of C. gattii to study how selection is acting on key virulence genes within different lineages.This article is part of the themed issue 'Tackling emerging fungal threats to animal health, food security and ecosystem resilience'.

Novel cell-based in vitro screen to identify small-molecule inhibitors against intracellular replication of Cryptococcus neoformans in macrophages.

The fungal pathogen Cryptococcus neoformans poses a major threat to immunocompromised patients and is a leading killer of human immunodeficiency virus (HIV)-infected patients worldwide. Cryptococci are known to manipulate host macrophages and can either remain latent or proliferate intracellularly within the host phagocyte, a favourable niche that also renders them relatively insensitive to antifungal agents. Here we report an attempt to address this limitation by using a fluorescence-based drug screening method to identify potential inhibitors of intracellular proliferation of C. neoformans. The Prestwick Chemical Library(®) of FDA-approved small molecules was screened for compounds that limit the intracellular replication of a fluorescently-tagged C. neoformans reference strain (H99-GFP) in macrophages. Preliminary screening revealed 19 of 1200 compounds that could significantly reduce intracellular growth of the pathogen. Secondary screening and host cell cytotoxicity assays highlighted fendiline hydrochloride as a potential drug candidate for the development of future anticryptococcal therapies. Live cell imaging demonstrated that this Ca(2+) channel blocker strongly enhanced phagosome maturation in macrophages leading to improved fungal killing and reduced intracellular replication. Whilst the relatively high dose of fendiline hydrochloride required renders it unfit for clinical deployment against cryptococcosis, this study highlights a novel approach for identifying new lead compounds and unravels a pharmacologically promising scaffold towards the development of novel antifungal therapies for this neglected disease.

Dos aislamientos de Salmonella Infantis multirresistentes se comportan como hipoinvasivos pero con elevada proliferación intracelular

En este trabajo se investigó la presencia de determinantes característicos de plásmidos de virulencia en dos aislamientos clínicos de Salmonella Infantis portadores de plásmidos de multirresistencia. Además, se estudió la capacidad de invasión y proliferación en células eucariotas no fagocíticas. Ninguno de los aislamientos de S. Infantis mostró los determinantes genéticos que caracterizan a los plásmidos de virulencia para este género (operón spv). Los ensayos de invasión sobre líneas celulares eucariotas mostraron que los aislamientos de S. Infantis presentan una capacidad de invasión disminuida pero persisten y proliferan en el citoplasma, independientemente de utilizar una línea celular permisiva (HeLa) o no permisiva (NRK) para tal fin. Finalmente, no se observaron indicios microscópicos que podrían hacer sospechar un efecto bactericida de estas líneas celulares sobre los aislamientos estudiados.

Two multidrug-resistant Salmonella Infantis isolates behave like hypo-invasive strains but have high intracellular proliferation. In this work, plasmid-encoded virulence factors in two Salmonella Infantis isolates carrying multiresistance plasmids were investigated. In addition, their invasion and proliferative ability in non-phagocytic cells was studied. None of them showed the typical determinants of virulence plasmids (spv operon). The invasion assays of S. Infantis isolates on eukaryotic cells showed a decreased ability to Invade but they remained and proliferated In the cytoplasm regardless of having used a permissive (HeLa) or non-permissive (NRK) cell line. Finally, there was no microscopic evidence suggesting a bactericidal effect of these eukaryotic cell lines on the Isolates tested.