Daniela Scribano

Outer Membrane Protein A (OmpA): A New Player in Shigella flexneri Protrusion Formation and Inter-Cellular Spreading

Outer membrane protein A (OmpA) is a multifaceted predominant outer membrane protein of Escherichia coli and other Enterobacteriaceae whose role in the pathogenesis of various bacterial infections has recently been recognized. Here, the role of OmpA on the virulence of Shigella flexneri has been investigated. An ompA mutant of wild-type S. flexneri 5a strain M90T was constructed (strain HND92) and it was shown to be severely impaired in cell-to-cell spreading since it failed to plaque on HeLa cell monolayers. The lack of OmpA significantly reduced the levels of IcsA while the levels of cell associated and released IcsP-cleaved 95 kDa amino-terminal portion of the mature protein were similar. Nevertheless, the ompA mutant displayed IcsA exposed across the entire bacterial surface. Surprisingly, the ompA mutant produced proper F-actin comet tails, indicating that the aberrant IcsA exposition at bacterial lateral surface did not affect proper activation of actin-nucleating proteins, suggesting that the absence of OmpA likely unmasks mature or cell associated IcsA at bacterial lateral surface. Moreover, the ompA mutant was able to invade and to multiply within HeLa cell monolayers, although internalized bacteria were found to be entrapped within the host cell cytoplasm. We found that the ompA mutant produced significantly less protrusions than the wild-type strain, indicating that this defect could be responsible of its inability to plaque. Although we could not definitely rule out that the ompA mutation might exert pleiotropic effects on other S. flexneri genes, complementation of the ompA mutation with a recombinant plasmid carrying the S. flexneri ompA gene clearly indicated that a functional OmpA protein is required and sufficient for proper IcsA exposition, plaque and protrusion formation. Moreover, an independent ompA mutant was generated. Since we found that both mutants displayed identical virulence profile, these results further supported the findings presented in this study.

Polar Localization of PhoN2, a Periplasmic Virulence-Associated Factor of Shigella flexneri, Is Required for Proper IcsA Exposition at the Old Bacterial Pole

Proper protein localization is critical for bacterial virulence. PhoN2 is a virulence-associated ATP-diphosphohydrolase (apyrase) involved in IcsA-mediated actin-based motility of S. flexneri. Herein, by analyzing a ΔphoN2 mutant of the S. flexneri strain M90T and by generating phoN2::HA fusions, we show that PhoN2, is a periplasmic protein that strictly localizes at the bacterial poles, with a strong preference for the old pole, the pole where IcsA is exposed, and that it is required for proper IcsA exposition. PhoN2-HA was found to be polarly localized both when phoN2::HA was ectopically expressed in a Escherichia coli K-12 strain and in a S. flexneri virulence plasmid-cured mutant, indicating a conserved mechanism of PhoN2 polar delivery across species and that neither IcsA nor the expression of other virulence-plasmid encoded genes are involved in this process. To assess whether PhoN2 and IcsA may interact, two-hybrid and cross-linking experiments were performed. While no evidence was found of a PhoN2-IcsA interaction, unexpectedly the outer membrane protein A (OmpA) was shown to bind PhoN2-HA through its periplasmic-exposed C-terminal domain. Therefore, to identify PhoN2 domains involved in its periplasmic polar delivery as well as in the interaction with OmpA, a deletion and a set of specific amino acid substitutions were generated. Analysis of these mutants indicated that neither the 183PAPAP187 motif of OmpA, nor the N-terminal polyproline 43PPPP46 motif and the Y155 residue of PhoN2 are involved in this interaction while P45, P46 and Y155 residues were found to be critical for the correct folding and stability of the protein. The relative rapid degradation of these amino acid-substituted recombinant proteins was found to be due to unknown S. flexneri-specific protease(s). A model depicting how the PhoN2-OmpA interaction may contribute to proper polar IcsA exposition in S. flexneri is presented.

T Follicular Helper Cells Promote a Beneficial Gut Ecosystem for Host Metabolic Homeostasis by sensing Microbiota-Derived Extracellular ATP.

Summary The ATP-gated ionotropic P2X7 receptor regulates T follicular helper (Tfh) cell abundance in the Peyer’s patches (PPs) of the small intestine; deletion of P2rx7, encoding for P2X7, in Tfh cells results in enhanced IgA secretion and binding to commensal bacteria. Here, we show that Tfh cell activity is important for generating a diverse bacterial community in the gut and that sensing of microbiota-derived extracellular ATP via P2X7 promotes the generation of a proficient gut ecosystem for metabolic homeostasis. The results of this study indicate that Tfh cells play a role in host-microbiota mutualism beyond protecting the intestinal mucosa by induction of affinity-matured IgA and suggest that extracellular ATP constitutes an inter-kingdom signaling molecule important for selecting a beneficial microbial community for the host via P2X7-mediated regulation of B cell help.

The Shigella flexneri OspB effector: an early immunomodulator

Through the action of the type three secretion system (T3SS) Shigella flexneri delivers several effectors into host cells to promote cellular invasion, multiplication and to exploit host-cell signaling pathways to modulate the host innate immune response. Although much progress has been made in the understanding of many type III effectors, the molecular and cellular mechanism of the OspB effector is still poorly characterized. In this study we present new evidence that better elucidates the role of OspB as pro-inflammatory factor at very early stages of infection. Indeed, we demonstrate that, during the first hour of infection, OspB is required for full activation of ERK1/2 and p38 MAPKs and the cytosolic phospholipase A(2) (cPLA(2)). Activation of cPLA(2) ultimately leads to the production and secretion of PMN chemoattractant metabolite(s) uncoupled with release of IL-8. Moreover, we also present evidence that OspB is required for the development of the full and promptly inflammatory reaction characteristic of S. flexneri wild-type infection in vivo. Based on OspB and OspF similarity (both effectors share similar transcription regulation, temporal secretion into host cells and nuclear localization) we hypothesized that OspB and OspF effectors may form a pair aimed at modulating the host cell response throughout the infection process, with opposite effects. A model is presented to illustrate how OspB activity would promote S. flexneri invasion and bacterial dissemination at early critical phases of infection.

First case report of invasive pseudoterranoviasis in Italy

Members of the genera Anisakis and Pseudoterranova are the main causative agents of human anisakidosis: the disease is worldwide distributed, with major impact in countries with a large consumption of raw fish. Because of unspecific symptoms and limited diagnostic tools, incidence and burden of disease are probably underestimated. In Italy, all human infestations where the etiological agent has been properly identified, have been associated to the parasitic species Anisakis pegreffii, the most frequent anisakid in the Mediterranean area. Here, an invasive human case of pseudoterranoviasis is described for the first time in Italy: in 2015, a woman was found infected during a colonoscopy scheduled after the occurrence of nonspecific clinical symptoms. The nematode was found penetrating the ascending colon. The identification was performed by sequencing the mitochondrial region cox2 and by comparison to GenBank retrieved material using the BLAST search tool. The sample showed a 99% identity with Pseudoterranova decipiens sensu stricto. The record underlines the potential risk due to the consumption of raw or undercooked imported fishes.

Increased Prevalence of Human Polyomavirus JC Viruria in Chronic Inflammatory Rheumatic Diseases Patients in Treatment with Anti-TNF α: A 18 Month Follow-Up Study

Chronic inflammatory rheumatic diseases (CIRDs) are immune-mediated pathologies involving joints. To date, TNFα-blocking agents administration is the most promising therapy, although these treatments are associated with an increased Polyomavirus JC (JCPyV) reactivation, the etiological agent of the Progressive Multifocal Leukoencephalopathy (PML). The aim of this study was the recruitment and the analysis of a CIRDs cohort in order to investigate a possible correlation between JCPyV presence and the influence of anti-TNF-α agents on viral loads. Blood and urine samples were collected from 34 CIRDs subjects prior the first anti-TNF-α infusion (T0) and after 3 (T3), 6 (T6), 12 (T12), and 18 (T18) months. Results showed persistent JC viruria significantly higher than JC viremia throughout the 18 month follow-up study (p = 0.002). In JCPyV positive samples, the non-coding control region (NCCR) was analyzed. Results evidenced archetypal structures (type II-S) in all isolates with the exception of a sequence isolated from a plasma sample, that corresponds to the type II-R found in PML subjects. Finally, the viral protein 1 (VP1) genotyping was performed and results showed the prevalence of the European genotypes 1A, 1B, and 4. Since only few studies have been carried out to understand whether there is a PML risk in CIRDs population infected by JCPyV, this study contributes to enrich literature insight on JCPyV biology in this cluster. Further investigations are necessary in order to recognize the real impact of biologics on JCPyV life cycle and to identify possible and specific viral variants related to increased virulence in CIRDs patients.

Efficient propagation of archetype JC polyomavirus in COS-7 cells: evaluation of rearrangements within the NCCR structural organization after transfection

John Cunningham virus (JCPyV) is an ubiquitous human pathogen that causes disease in immunocompromised patients. The JCPyV genome is composed of an early region and a late region, which are physically separated by the non-coding control region (NCCR). The DNA sequence of the NCCR distinguishes two forms of JCPyV, the designated archetype and the prototype, which resulted from a rearrangement of the archetype sequence. To date, the cell culture systems for propagating JCPyV archetype have been very limited in their availability and robustness. Prior to this study, it was demonstrated that JCPyV archetype DNA replicates in COS-7 simian kidney cells expressing SV40 TAg and COS-7 cells expressing HIV-1 Tat. Based on these observations, the present study was conducted to reproduce an in vitro model in COS-7 cells transfected with the JCPyV archetype strain in order to study JCPyV DNA replication and analyze NCCR rearrangements during the viral life cycle. The efficiency of JCPyV replication was evaluated by quantitative PCR (Q-PCR) and by hemagglutination (HA) assay after transfection. In parallel, sequence analysis of JCPyV NCCR was performed. JCPyV efficiently replicated in kidney-derived COS-7 cells, as demonstrated by a progressive increase in viral load and virion particle production after transfection. The archetypal structure of NCCR was maintained during the viral cycle, but two characteristic point mutations were detected 28 days after transfection. This model is a useful tool for analyzing NCCR rearrangements during in vitro replication in cells that are sites of viral persistence, such as tubular epithelial cells of the kidney.

Genetic diversity, phylogroup distribution and virulence gene profile of pks positive Escherichia coli colonizing human intestinal polyps

Some Escherichia coli strains of phylogroup B2 harbor a (pks) pathogenicity island that encodes a polyketide-peptide genotoxin called colibactin. It causes DNA double-strand breaks and megalocytosis in eukaryotic cells and it may contribute to cancer development. Study of bacterial community that colonizes the adenomatous polyp lesion, defined as precancerous lesions, could be helpful to assess if such pathogenic bacteria possess a role in the polyp progression to cancer. In this cross-sectional study, a total of 1500 E. coli isolates were obtained from biopsies of patients presenting adenomatous colon polyps, the normal tissues adjacent to the polyp lesion and patients presenting normal mucosa. pks island frequency, phylogenetic grouping, fingerprint genotyping, and virulence gene features of pks positive (pks+) E. coli isolates were performed. We found pks+E. coli strongly colonize two patients presenting polypoid lesions and none were identified in patients presenting normal mucosa. Predominant phylogroups among pks+E. coli isolates were B2, followed by D. Clustering based on fragment profiles of composite analysis, typed the pks+ isolates into 5 major clusters (I-V) and 17 sub-clusters, demonstrating a high level of genetic diversity among them. The most prevalent virulence genes were fimH and fyuA (100%), followed by vat (92%), hra and papA (69%), ibeA (28%), and hlyA (25%). Our results revealed that pks+E. coli can colonize the precancerous lesions, with a high distribution in both the polyp lesions and in normal tissues adjacent to the lesion. The high differences in fingerprinting patterns obtained indicate that pks+E. coli strains were genetically diverse, possibly allowing them to more easily adapt to environmental variations.

COS-7-based model: methodological approach to study John Cunningham virus replication cycle

John Cunningham virus (JCV) is a human neurotropic polyomavirus whose replication in the Central Nervous System (SNC) induces the fatal demyelinating disease, progressive multifocal leukoencephalopathy (PML). JCV propagation and PML investigation have been severely hampered by the lack of an animal model and cell culture systems to propagate JCV have been very limited in their availability and robustness. We previously confirmed that JCV CY strain efficiently replicated in COS-7 cells as demonstrated by the progressive increase of viral load by quantitative PCR (Q-PCR) during the time of transfection and that archetypal regulatory structure was maintained, although two characteristic point mutations were detected during the viral cycle. This short report is an important extension of our previous efforts in defining our reliable model culture system able to support a productive JCV infection.Supernatants collected from transfected cells have been used to infect freshly seeded COS-7 cell line. An infectious viral progeny was obtained as confirmed by Western blot and immunofluorescence assay. During infection, the archetype regulatory region was conserved.Importantly, in this study we developed an improved culture system to obtain a large scale production of JC virus in order to study the genetic features, the biology and the pathogenic mechanisms of JC virus that induce PML.

Cutaneous candidiasis caused by Candida albicans in a young non-immunosuppressed patient: an unusual presentation:

Candidiasis is a fungal infection caused by yeasts that belong to the genus Candida. There are over 20 species of Candida yeasts that can cause infection in humans, the most common of which is Candida albicans. Candida yeasts normally reside in the intestinal tract and can be found on mucous membranes and skin without causing infection. However, under immunocompromised conditions, Candida can cause significant infections in susceptible patients. Herein, we report a peculiar presentation of a C. albicans cutaneous infection in an immunocompetent young subject. This case widens our knowledge on the C. albicans infections both in terms of host susceptibility and cutaneous manifestations.