Stefano Rocca

PE_PGRS30 is required for the full virulence of Mycobacterium tuberculosis

The role and function of PE_PGRS proteins of Mycobacterium tuberculosis (Mtb) remains elusive. In this study for the first time, Mtb isogenic mutants missing selected PE_PGRSs were used to investigate their role in the pathogenesis of tuberculosis (TB). We demonstrate that the MtbΔPE_PGRS30 mutant was impaired in its ability to colonize lung tissue and to cause tissue damage, specifically during the chronic steps of infection. Inactivation of PE_PGRS30 resulted in an attenuated phenotype in murine and human macrophages due to the inability of the Mtb mutant to inhibit phagosome–lysosome fusion. Using a series of functional deletion mutants of PE_PGRS30 to complement MtbΔPE_PGRS30, we show that the unique C‐terminal domain of the protein is not required for the full virulence. Interestingly, when Mycobacterium smegmatis recombinant strain expressing PE_PGRS30 was used to infect macrophages or mice in vivo, we observed enhanced cytotoxicity and cell death, and this effect was dependent upon the PGRS domain of the protein.Taken together these results indicate that PE_PGRS30 is necessary for the full virulence of Mtb and sufficient to induce cell death in host cells by the otherwise non‐pathogenic species M. smegmatis, clearly demonstrating that PE_PGRS30 is an Mtb virulence factor.

2-D PAGE and MS analysis of proteins from formalin-fixed, paraffin-embedded tissues

In the past decade, encouraging results have been obtained in extraction and analysis of proteins from formalin‐fixed, paraffin‐embedded (FFPE) tissues. However, 2‐D PAGE protein maps with satisfactory proteomic information and comparability to fresh tissues have never been described to date. In the present study, we report 2‐D PAGE separation and MS identification of full‐length proteins extracted from FFPE skeletal muscle tissue. The 2‐D protein profiles obtained from FFPE tissues could be matched to those achieved from frozen tissues replicates. Up to 250 spots were clearly detected in 2‐D maps of proteins from FFPE tissue following standard mass‐compatible silver staining. Protein spots from both FFPE and frozen tissue 2‐D gels were excised, subjected to in situ hydrolysis, and identified by MS analysis. Matched spots produced matched protein identifications. Moreover, 2‐D protein maps from FFPE tissues were successfully subjected to Western immunoblotting, producing comparable results to fresh‐frozen tissues. In conclusion, this study provides evidence that, when adequately extracted, full‐length proteins from FFPE tissues might be suitable to 2‐D PAGE‐MS analysis, allowing differential proteomic studies on the vast existing archives of healthy and pathological‐fixed tissues.

Molecular Investigation and Phylogeny of Anaplasma spp. in Mediterranean Ruminants Reveal the Presence of Neutrophil-Tropic Strains Closely Related to A. platys

ABSTRACT Few data are available on the prevalence and molecular typing of species belonging to the genus Anaplasma in Mediterranean ruminants. In this study, PCR analysis and sequencing of both 16S rRNA and groEL genes were combined to investigate the presence, prevalence, and molecular traits of Anaplasma spp. in ruminants sampled on the Island of Sardinia, chosen as a subtropical representative area. The results demonstrate a high prevalence of Anaplasma spp. in ruminants, with animals infected by at least four of six Anaplasma species (Anaplasma marginale, A. bovis, A. ovis, and A. phagocytophilum). Moreover, ruminants host a number of neutrophil-tropic strains genetically closely related to the canine pathogen A. platys. The high Anaplasma spp. prevalence and the identification of as-yet-unclassified neutrophil-tropic strains raise concerns about the specificity of serological tests routinely used in ruminants and provide additional background for reconstructing the evolutionary history of species genetically related to A. phagocytophilum.

The hbhA Gene of Mycobacterium tuberculosis Is Specifically Upregulated in the Lungs but Not in the Spleens of Aerogenically Infected Mice

ABSTRACT We report that hbhA is differentially regulated during Mycobacterium tuberculosis infection. Upregulation was observed in epithelial cell infection but not in macrophage infection and in the lungs but not in the spleens of infected mice, and it was greater during the early steps of infection, when bacilli disseminate from the site of primary infection.

Surface expression of MPT64 as a fusion with the PE domain of PE_PGRS33 enhances Mycobacterium bovis BCG protective activity against Mycobacterium tuberculosis in mice.

ABSTRACT To improve the current vaccine against tuberculosis, a recombinant strain of Mycobacterium bovis bacillus Calmette-Guérin (rBCG) expressing a Mycobacterium tuberculosis vaccine candidate antigen (MPT64) in strong association with the mycobacterial cell wall was developed. To deliver the candidate antigen on the surface, we fused the mpt64 gene to the sequence encoding the PE domain of the PE_PGRS33 protein of M. tuberculosis (to create strain HPE-ΔMPT64-BCG), which we have previously shown to transport proteins to the bacterial surface. In a series of protection experiments in the mouse model of tuberculosis, we showed that (i) immunization of mice with HPE-ΔMPT64-BCG provides levels of protection significantly higher than those afforded by the parental BCG strain, as assessed by bacterial colonization in lungs and spleens and by lung involvement (at both 28 and 70 days postchallenge), (ii) rBCG strains expressing MPT64 provide better protection than the parental BCG strain only when this antigen is surface expressed, and (iii) the HPE-ΔMPT64-BCG-induced MPT64-specific T cell repertoire when characterized by β chain variable region-β chain joining region (BV-BJ) spectratyping indicates that protection is correlated with the ability to recruit gamma interferon (IFN-γ)-secreting T cells carrying the BV8.3-BJ1.5 (172 bp) shared rearrangement. These results demonstrate that HPE-ΔMPT64-BCG is one of the most effective new vaccines tested so far in the mouse model of tuberculosis and underscore the impact of antigen cellular localization on the induction of the specific immune response induced by rBCG.

Application of 2-D DIGE to formalin-fixed, paraffin-embedded tissues

The ability to investigate the proteome of formalin‐fixed, paraffin‐embedded (FFPE) tissues can be considered a major recent achievement in the field of clinical proteomics. However, gel‐based approaches to the investigation of FFPE tissue proteomes have lagged behind, mainly because of insufficient quality of full‐length protein extracts. Here, the 2‐D DIGE technology was investigated for applicability to FFPE proteins, for internal reproducibility among replicate FFPE extracts, and for comparability between FFPE and fresh‐frozen tissue profiles. The 2‐D DIGE patterns obtained upon labeling and electrophoresis of replicate FFPE tissue extracts were highly reproducible, with satisfactory resolution and complexity. Moreover, the implementation of DIGE enabled to highlight and characterize the consistent differences found in the FFPE profiles compared with fresh‐frozen profiles, represented by an acidic shift, directly correlated to the protein pI value, and by a reduction in spot signal intensity, directly correlated to molecular weight and percentage of lysine residues. Being constantly and reproducibly present in all FFPE tissue extract replicates at similar extents, these modifications do not appear to hinder the comparative analysis of FFPE tissue extracts by 2‐D DIGE, opening the way to its application for the differential proteomic investigation of archival tissue repositories.

Sheep embryonic stem-like cells transplanted in full-thickness cartilage defects.

Articular cartilage regeneration is limited. Embryonic stem (ES) cell lines provide a source of totipotent cells for regenerating cartilage. Anatomical, biomechanical, physiological and immunological similarities between humans and sheep make this animal an optimal experimental model. This study examines the repair process of articular cartilage in sheep after transplantation of ES‐like cells isolated from inner cell masses (ICMs) derived from in vitro‐produced (IVP) vitrified embryos. Thirty‐five ES‐like colonies from 40 IVP embryos, positive for stage‐specific embryonic antigens (SSEAs), were pooled in groups of two or three, embedded in fibrin glue and transplanted into osteochondral defects in the medial femoral condyles of 14 ewes. Empty defect (ED) and cell‐free glue (G) in the controlateral stifle joint served as controls. The Y gene sequence was used to detect ES‐like cells in the repair tissue by in situ hybridization (ISH). Two ewes were euthanized at 1 month post‐operatively, three each at 2 and 6 months and four at 12 months. Repairing tissue was examined by biomechanical, macroscopic, histological, immunohistochemical (collagen type II) and ISH assays. Scores of all treatments showed no statistical significant differences among treatment groups at a given time period, although ES‐like grafts showed a tendency toward a better healing process. ISH was positive in all ES‐like specimens. This study demonstrates that ES‐like cells transplanted into cartilage defects stimulate the repair process to promote better organization and tissue bulk. However, the small number of cells applied and the short interval between surgery and euthanasia might have negatively affected the results. Copyright

Production and release of antimicrobial and immune defense proteins by mammary epithelial cells following Streptococcus uberis infection of sheep.

ABSTRACT Investigating the innate immune response mediators released in milk has manifold implications, spanning from elucidation of the role played by mammary epithelial cells (MECs) in fighting microbial infections to the discovery of novel diagnostic markers for monitoring udder health in dairy animals. Here, we investigated the mammary gland response following a two-step experimental infection of lactating sheep with the mastitis-associated bacterium Streptococcus uberis. The establishment of infection was confirmed both clinically and by molecular methods, including PCR and fluorescent in situ hybridization of mammary tissues. Proteomic investigation of the milk fat globule (MFG), a complex vesicle released by lactating MECs, enabled detection of enrichment of several proteins involved in inflammation, chemotaxis of immune cells, and antimicrobial defense, including cathelicidins and calprotectin (S100A8/S100A9), in infected animals, suggesting the consistent involvement of MECs in the innate immune response to pathogens. The ability of MECs to produce and release antimicrobial and immune defense proteins was then demonstrated by immunohistochemistry and confocal immunomicroscopy of cathelicidin and the calprotectin subunit S100A9 on mammary tissues. The time course of their release in milk was also assessed by Western immunoblotting along the course of the experimental infection, revealing the rapid increase of these proteins in the MFG fraction in response to the presence of bacteria. Our results support an active role of MECs in the innate immune response of the mammary gland and provide new potential for the development of novel and more sensitive tools for monitoring mastitis in dairy animals.

Neutrophil extracellular traps in sheep mastitis

Neutrophil extracellular traps (NETs) are structures composed of DNA, histones, and antimicrobial proteins that are released extracellularly by neutrophils and other immune cells as a means for trapping and killing invading pathogens. Here, we describe NET formation in milk and in mammary alveoli of mastitic sheep, and provide a dataset of proteins found in association to these structures. Nucleic acid staining, immunomicroscopy and fluorescent in-situ hybridization of mastitic mammary tissue from sheep infected with Streptococcus uberis demonstrated the presence of extranuclear DNA colocalizing with antimicrobial proteins, histones, and bacteria. Then, proteomic analysis by LTQ-Orbitrap Velos mass spectrometry provided detailed information on protein abundance changes occurring in milk upon infection. As a result, 1095 unique proteins were identified, of which 287 being significantly more abundant in mastitic milk. Upon protein ontology classification, the most represented localization classes for upregulated proteins were the cytoplasmic granule, the nucleus, and the mitochondrion, while function classes were mostly related to immune defence and inflammation pathways. All known NET markers were massively increased, including histones, granule proteases, and antimicrobial proteins. Of note was the detection of protein arginine deiminases (PAD3 and PAD4). These enzymes are responsible for citrullination, the post-translational modification that is known to trigger NET formation by inducing chromatin decondensation and extracellular release of NETs. As a further observation, citrullinated residues were detected by tandem mass spectrometry in histones of samples from mastitic animals. In conclusion, this work provides novel microscopic and proteomic information on NETs formed in vivo in the mammary gland, and reports the most complete database of proteins increased in milk upon bacterial mastitis.

Impact of fixation time on GeLC-MS/MS proteomic profiling of formalin-fixed, paraffin-embedded tissues.

Formalin-fixed, paraffin-embedded (FFPE) tissue banks represent an invaluable resource for biomarker discovery. Recently, the combination of full-length protein extraction, GeLC-MS/MS analysis, and spectral counting quantification has been successfully applied to mine proteomic information from these tissues. However, several sources of variability affect these samples; among these, the duration of the fixation process is one of the most important and most easily controllable ones. To assess its influence on quality of GeLC-MS/MS data, the impact of fixation time on efficiency of full-length protein extraction efficiency and on quality of label-free quantitative data was evaluated. As a result, although proteins were successfully extracted from FFPE liver samples fixed for up to eight days, fixation time appeared to negatively influence both protein extraction yield and GeLC-MS/MS quantitative proteomic data. Particularly, MS identification efficiency decreased with increasing fixation times. Moreover, amino acid modifications putatively induced by formaldehyde were detected and characterized. These results demonstrate that proteomic information can be achieved also from tissue samples fixed for relatively long times, but suggest that variations in fixation time need to be carefully taken into account when performing proteomic biomarker discovery studies on fixed tissue archives.