Molecular variability of Meningococcal antigens in carriage and disease strains and in other Neisseria species

This PhD thesis is focused on the study of the molecular variability of some specific proteins, part of the outer membrane of the pathogen Neisseria meningitidis, and described as protective antigens and important virulence factors. These antigens have been employed as components of the vaccine developed by Novartis Vaccines against N. meningitidis of serogroup B, and their variability in the meningococcal population is a key aspect when the effect of the vaccine is evaluated. The PhD project has led to complete three major studies described in three different manuscritps, of which two have been published and the third is in preparation. The thesis is structured in three main chapters, each of them dedicated to the three studies. The first, described in Chapter 1, is specifically dedicated to the analysis of the molecular conservation of meningococcal antigens in the genomes of all species classified in the genus Neisseria (Conservation of Meningococcal Antigens in the Genus Neisseria. A. Muzzi et al.. 2013. mBio 4 (3)). The second study, described in Chapter 2, focuses on the analysis of the presence and conservation of the antigens in a panel of bacterial isolates obtained from cases of the disease and from healthy individuals, and collected in the same year and in the same geographical area (Conservation of fHbp, NadA, and NHBA in carrier and pathogenic isolates of Neisseria meningitidis collected in the Czech Republic in 1993. A. Muzzi et al.. Manuscript in preparation). Finally, Chapter 3 describes the molecular features of the antigens in a panel of bacterial isolates collected over a period of 50 years, and representatives of the epidemiological history of meningococcal disease in the Netherlands (An Analysis of the Sequence Variability of Meningococcal fHbp, NadA and NHBA over a 50-Year Period in the Netherlands. S. Bambini et al.. 2013. PloS one e65043).

Cell death mechanisms triggered by monoclonal antibodies against CD99 in Ewing sarcoma: Cross-talk between MDM2, IGF-1R and Ras/MAPK

CD99 is a 32 kDa transmembrane protein whose high expression characterizes Ewing sarcoma (ES), a very aggressive pediatric bone tumor. In addition to its diagnostic value, CD99 has therapeutic potential since it leads to rapid and massive ES cell death when engaged with specific antibodies. Here a novel mechanism of cell death triggered via CD99 is shown, leading, ultimately, to the appearance of macropinocytotic vescicles. Anti-CD99 mAb 0662 induces MDM2 ubiquitination and degradation, which causes not only a p53 reactivation but also the IGF-1R induction and its subsequent internalization; CD99 results internalized together with IGF-1R inside endosomes, but then the two molecules display a different sorting: CD99 is degraded, while IGF-1R is recycled on the surface, causing, as a final step, the up-regulation of RAS-MAPK. High-expressing CD99 mesenchymal stem cells show mild Ras induction but no p53 activation and escape cell death, but in presence of EWS/FLI1 mesenchymal stem cells expressing CD99 show a stronger Ras induction and a p53 reactivation, leading to a significant cell death rate. We propose that CD99 triggering in a EWS/FLI1-driven oncogenetic context creates a synergy between RAS upregulation and p53 activation in ES cells, leading to cell death. Moreover, our data rule out possible concerns on toxicity related to the broad CD99 expression in normal tissues and provide the rationale for the therapeutic use of anti-CD99 MAbs in the clinic.

Staphylococcus aureus bones and joints infections: in vivo studies and host immune response

Abstract The aim of this work was the development of a murine model of septic arthrosynovitis and osteomyelitis caused by Staphylococcus aureus, which could mimic the natural disease occurring in humans and which could be suitable for testing preventive and therapeutic interventions. This model could be particularly useful since S. aureus-mediated joints and bones infections are relevant in humans, both in terms of frequency and severity. Our attention focused in tracking bacterial infiltration in joints and bones over time using different microbiological and hystopathological tools, which allowed us to have a complete overview of the situation and to evaluate the immunological actions undertaken by the host to contain or eradicate the bacterial infection. Antibodies and cytokines profiles, as well as recruitment of host immune cells at joints of immunized and infected mice were therefore monitored for a time period that allowed us to study both the acute and the chronic phases of the disease in situ. Finally the Novartis vaccine formulation proposed against S. aureus infections was tested for its capacity to protect immunized mice from joints infections, and the preventive immunization was compared to a standard antibiotic prophylaxis. The availability of powerful tools to study specific bacterial-mediated diseases is nowadays an important requirement for the scientific community to shed light on the complex interactions between host and pathogens and to test treatments for preventing or contrasting infections. We believe that our work significantly contributes to the overall knowledge in the field of S. aureus-dependent pathologies, opening the possibility for further investigations in several fields of study.

Peptidi autoassemblanti per la produzione di scaffold nell'ingegneria tissutale

L’argomento trattato in questo elaborato riguarda la natura e le applicazioni di una nuova classe di biomateriali: i peptidi auto-assemblanti. La perdita di funzione di un organo o di un tessuto rappresenta una problematica rilevante sia sotto il profilo clinico sia per i costi di gestione. I trapianti sono infatti tra le terapie più sofisticate e onerose economicamente, complicate da altri aspetti quali una strutturale insufficienza di donatori e la necessità che i soggetti trapiantati vengano sottoposti cronicamente a regimi terapeutici immunosoppressivi che aumentano eventuali effetti collaterali. La terapia sostitutiva basata su organi artificiali è invece gravata dalla durata limitata dei dispositivi, nonchè da un non trascurabile rischio infettivo. La medicina rigenerativa, che sembra essere una soluzione adeguata per ovviare a tutte queste problematiche, è un settore emergente che combina aspetti della medicina, della biologia cellulare e molecolare, della scienza dei materiali e dell’ingegneria al fine di rigenerare, riparare o sostituire i tessuti danneggiati. In questo panorama, il ruolo dei biomateriali sta diventando sempre più importante grazie alla loro varietà e alle loro funzioni emergenti. Tra i biomateriali innovativi più promettenti troviamo i peptidi auto-assemblanti. Dopo un'introduzione sui principi dell'ingegneria tissutale, la tesi si focalizza sui peptidi auto-assemblanti e sulle loro applicazioni in campo biomedico, ponendo l'attenzione, in particolar modo, sulla realizzazione di scaffold per la rigenerazione del tessuto osseo, cardiaco, cartilagineo e nervoso, e sulla loro applicazione per il rilascio controllato di farmaci.

Interactions between the gut microbiota, short-chain fatty acids and the immune system in pediatric patients undergoing hematopoietic stem cell transplantation

The gut microbiota (GM) is essential for human health and contributes to several diseases; indeed it can be considered an extension of the self and, together with the genetic makeup, determines the physiology of an organism. In this thesis has been studied the peripheral immune system reconstitution in pediatric patients undergoing allogeneic hematopoietic stem cell transplantation (aHSCT) in the early phase; in parallel, have been also explored the gut microbiota variations as one of the of primary factors in governing the fate of the immunological recovery, predisposing or protecting from complications such as the onset of acute graft-versus-host disease (GvHD). Has been demonstrated, to our knowledge for the first time, that aHSCT in pediatric patients is associated to a profound modification of the GM ecosystem with a disruption of its mutualistic asset. aGvHD and non-aGvHD subjects showed differences in the process of GM recovery, in members abundance of the phylum Bacteroidetes, and in propionate fecal concentration; the latter are higher in the pre-HSCT composition of non-GvHD subjects than GvHD ones. Short-chain fatty acids (SCFAs), such as acetate, butyrate and propionate, are end-products of microbial fermentation of macronutrients and distribute systemically from the gut to blood. For this reason, has been studied their effect in vitro on human DCs, the key regulators of our immune system and the main player of aGvHD onset. Has been observed that propionate and, particularly, butyrate show a strong and direct immunomodulatory activity on DCs reducing inflammatory markers such as chemokines and interleukins. This study, with the needed caution, suggests that the pre-existing GM structure can be protective against aGvHD onset, exerting its protective role through SCFAs. They, indeed, may regulate cell traffic within secondary lymphoid tissues, influence T cell development during antigen recognition, and, thus, directly shape the immune system.

Structural and functional characterization of Group B Streptococcus pilus 2b

Group B Streptococcus (GBS) is a Gram-positive human pathogen representing one of the most common causes of life-threatening bacterial infections such as sepsis and meningitis in neonates. Covalently polymerized pilus-like structures have been discovered in GBS as important virulence factors as well as vaccine candidates. Pili are protein polymers forming long and thin filamentous structures protruding from bacterial cells, mediating adhesion and colonization to host cells. Gram-positive bacteria, including GBS, build pili on their cell surface via a class C sortase-catalyzed transpeptidation mechanism from pilin protein substrates that are the backbone protein forming the pilus shaft and two ancillary proteins. Also the cell-wall anchoring of the pilus polymers made of covalently linked pilin subunits is mediated by a sortase enzyme. GBS expresses three structurally distinct pilus types (type 1, 2a and 2b). Although the mechanisms of assembly and cell wall anchoring of GBS types 1 and 2a pili have been investigated, those of pilus 2b are not understood until now. Pilus 2b is frequently found in ST-17 strains that are mostly associated with meningitis and high mortality rate especially in infants. In this work the assembly mechanism of GBS pilus type 2b has been elucidated by dissecting through genetic, biochemical and structural studies the role of the two pilus-associated sortases. The most significant findings show that pilus 2b assembly appears “non-canonical”, differing significantly from current pilus assembly models in Gram-positive pathogens. Only sortase-C1 is involved in pilin polymerization, while the sortase-C2 does not act as a pilin polymerase, but it is involved in cell-wall pilus anchoring. Our findings provide new insights into pili biogenesis in Gram-positive bacteria. Moreover, the role of this pilus type during host infection has been investigated. By using a mouse model of meningitis we demonstrated that type 2b pilus contributes to pathogenesis of meningitis in vivo.

Chemopreventive effects of eicosapentaenoic acid free fatty acid in a murine model of colitis-associated colorectal cancer

Inflammatory bowel diseases are associated with increased risk of developing colitis-associated colorectal cancer (CAC). Epidemiological data show that the consumption of ω-3 polyunsaturated fatty acids (ω-3 PUFAs) decreases the risk of sporadic colorectal cancer (CRC). Importantly, recent data have shown that eicosapentaenoic acid-free fatty acid (EPA-FFA) reduces polyps formation and growth in models of familial adenomatous polyposis. However, the effects of dietary EPA-FFA are unknown in CAC. We tested the effectiveness of substituting EPA-FFA, for other dietary fats, in preventing inflammation and cancer in the AOM-DSS model of CAC. The AOM-DSS protocols were designed to evaluate the effect of EPA-FFA on both initiation and promotion of carcinogenesis. We found that EPA-FFA diet strongly decreased tumor multiplicity, incidence and maximum tumor size in the promotion and initiation arms. Moreover EPA-FFA, in particular in the initiation arm, led to reduced cell proliferation and nuclear β-catenin expression, whilst it increased apoptosis. In both arms, EPA-FFA treatment led to increased membrane switch from ω-6 to ω-3 PUFAs and a concomitant reduction in PGE2 production. We observed no significant changes in intestinal inflammation between EPA-FFA treated arms and AOM-DSS controls. Importantly, we found that EPA-FFA treatment restored the loss of Notch signaling found in the AOM-DSS control, resulted in the enrichment of Lactobacillus species in the gut microbiota and led to tumor suppressor miR34-a induction. In conclusion, our data suggest that EPA-FFA is an effective chemopreventive agent in CAC.

Interactions of G-quadruplex binders and Topoisomerase I inhibitors in cancer cells

Top1-DNA cleavage complexes (Top1ccs) trigger an accumulation of antisense RNAPII transcripts specifically at active divergent CpG-island promoters in a replication independent and Top1 dependent manner, leading to transcription-dependent genome instability and altered transcription regulation. Using different cancer cell lines of colon and osteo origins, we show that they display different sensitivity to CPT and G4 binder that is independent from Top1 level. To look at the interactions between Top1 and G4, we show that co-treatment with G4 binders potentiate the cell cytotoxicity of CPT regardless of the treatment sequences. Potentiation is indicated by a reduced inhibition concentration (IC50) with a more profound cytotoxicity in CPT-resistant cell lines, HCT15 and U2OS, hence, indicating an interaction between Top1inhibitor and G4 binders. Moreover, computational analysis confirmed the present of G4 motifs in genes with CPT-induced antisense transcription. G4 motifs are present mostly 5000 bp upstream from transcription start site and notably lower in genes. Comparisons between genes with no antisense transcription and genes with antisense transcription show that G4 motifs in this region are notably lower in the genes with antisense transcripts. Since CPT increases negative supercoils at promoters of intermediate activity, the formation of G4 is also increased in CPT-treated cells. Suprisingly, formation of G4 is regulated in parallel to the transient stabilization of R-loops, indicating a role in response to CPT-induced stress. G4 formation is highly elevated in Pyridostatin treated cells, which previous study shows increased formation of γH2Ax foci. This effect is also seen in the CPT-resistant cell lines, HCT15, indicating that the formation is a general event in response to CPT. We also show that R-loop formation is greatly increased in Pyridostatin treated cells. In order to study the role of R-loops and G4 structures in Top1cc-dependant repair pathway, we inhibited tyrosyl-phosphodiestrase 1 (TDP-1) using a TDP-1 inhibitor.

Natural compounds Camptothecin and Triptolide: highly specific enzyme inhibitors and tools to dissect transcriptional functions

With this work I elucidated new and unexpected mechanisms of two strong and highly specific transcription inhibitors: Triptolide and Campthotecin. Triptolide (TPL) is a diterpene epoxide derived from the Chinese plant Trypterigium Wilfoordii Hook F. TPL inhibits the ATPase activity of XPB, a subunit of the general transcription factor TFIIH. In this thesis I found that degradation of Rbp1 (the largest subunit of RNA Polymerase II) caused by TPL treatments, is preceded by an hyperphosphorylation event at serine 5 of the carboxy-terminal domain (CTD) of Rbp1. This event is concomitant with a block of RNA Polymerase II at promoters of active genes. The enzyme responsible for Ser5 hyperphosphorylation event is CDK7. Notably, CDK7 downregulation rescued both Ser5 hyperphosphorylation and Rbp1 degradation triggered by TPL. Camptothecin (CPT), derived from the plant Camptotheca acuminata, specifically inhibits topoisomerase 1 (Top1). We first found that CPT induced antisense transcription at divergent CpG islands promoter. Interestingly, by immunofluorescence experiments, CPT was found to induce a burst of R loop structures (DNA/RNA hybrids) at nucleoli and mitochondria. We then decided to investigate the role of Top1 in R loop homeostasis through a short interfering RNA approach (RNAi). Using DNA/RNA immunoprecipitation techniques coupled to NGS I found that Top1 depletion induces an increase of R loops at a genome-wide level. We found that such increase occurs on the entire gene body. At a subset of loci R loops resulted particularly stressed after Top1 depletion: some of these genes showed the formation of new R loops structures, whereas other loci showed a reduction of R loops. Interestingly we found that new peaks usually appear at tandem or divergent genes in the entire gene body, while losses of R loop peaks seems to be a feature specific of 3’ end regions of convergent genes.

Exploring host-pathogen interactions through protein microarray. Large-scale protein microarray analysis revealed novel human receptors for the staphylococcal immune evasion protein FLIPr and for the neisserial adhesin NadA

Adhesion, immune evasion and invasion are key determinants during bacterial pathogenesis. Pathogenic bacteria possess a wide variety of surface exposed and secreted proteins which allow them to adhere to tissues, escape the immune system and spread throughout the human body. Therefore, extensive contacts between the human and the bacterial extracellular proteomes take place at the host-pathogen interface at the protein level. Recent researches emphasized the importance of a global and deeper understanding of the molecular mechanisms which underlie bacterial immune evasion and pathogenesis. Through the use of a large-scale, unbiased, protein microarray-based approach and of wide libraries of human and bacterial purified proteins, novel host-pathogen interactions were identified. This approach was first applied to Staphylococcus aureus, cause of a wide variety of diseases ranging from skin infections to endocarditis and sepsis. The screening led to the identification of several novel interactions between the human and the S. aureus extracellular proteomes. The interaction between the S. aureus immune evasion protein FLIPr (formyl-peptide receptor like-1 inhibitory protein) and the human complement component C1q, key players of the offense-defense fighting, was characterized using label-free techniques and functional assays. The same approach was also applied to Neisseria meningitidis, major cause of bacterial meningitis and fulminant sepsis worldwide. The screening led to the identification of several potential human receptors for the neisserial adhesin A (NadA), an important adhesion protein and key determinant of meningococcal interactions with the human host at various stages. The interaction between NadA and human LOX-1 (low-density oxidized lipoprotein receptor) was confirmed using label-free technologies and cell binding experiments in vitro. Taken together, these two examples provided concrete insights into S. aureus and N. meningitidis pathogenesis, and identified protein microarray coupled with appropriate validation methodologies as a powerful large scale tool for host-pathogen interactions studies.

Regulatory networks of Neisseria meningitidis and their implications for pathogenesis

Neisseria meningitidis, the leading cause of bacterial meningitis, can adapt to different host niches during human infection. Both transcriptional and post-transcriptional regulatory networks have been identified as playing a crucial role for bacterial stress responses and virulence. We investigated the N. meningitidis transcriptional landscape both by microarray and by RNA sequencing (RNAseq). Microarray analysis of N. meningitidis grown in the presence or absence of glucose allowed us to identify genes regulated by carbon source availability. In particular, we identified a glucose-responsive hexR-like transcriptional regulator in N. meningitidis. Deletion analysis showed that the hexR gene is accountable for a subset of the glucose-responsive regulation, and in vitro assays with the purified protein showed that HexR binds to the promoters of the central metabolic operons of meningococcus, by targeting a DNA region overlapping putative regulatory sequences. Our results indicate that HexR coordinates the central metabolism of meningococcus in response to the availability of glucose, and N. meningitidis strains lacking the hexR gene are also deficient in establishing successful bacteremia in a mouse model of infection. In parallel, RNAseq analysis of N. meningitidis cultured under standard or iron-limiting in vitro growth conditions allowed us to identify novel small non-coding RNAs (sRNAs) potentially involved in N. meningitidis regulatory networks. Manual curation of the RNAseq data generated a list of 51 sRNAs, 8 of which were validated by Northern blotting. Deletion of selected sRNAs caused attenuation of N. meningitidis infection in a murine model, leading to the identification of the first sRNAs influencing meningococcal bacteraemia. Furthermore, we describe the identification and initial characterization of a novel sRNA unique to meningococcus, closely associated to genes relevant for the intracellular survival of pathogenic Neisseriae. Taken together, our findings could help unravel the regulation of N. meningitidis adaptation to the host environment and its implications for pathogenesis.

Evaluation of 3D cell culture systems for host-pathogen interaction studies

Traditional cell culture models have limitations in extrapolating functional mechanisms that underlie strategies of microbial virulence. Indeed during the infection the pathogens adapt to different tissue-specific environmental factors. The development of in vitro models resembling human tissue physiology might allow the replacement of inaccurate or aberrant animal models. Three-dimensional (3D) cell culture systems are more reliable and more predictive models that can be used for the meaningful dissection of host–pathogen interactions. The lung and gut mucosae often represent the first site of exposure to pathogens and provide a physical barrier against their entry. Within this context, the tracheobronchial and small intestine tract were modelled by tissue engineering approach. The main work was focused on the development and the extensive characterization of a human organotypic airway model, based on a mechanically supported co-culture of normal primary cells. The regained morphological features, the retrieved environmental factors and the presence of specific epithelial subsets resembled the native tissue organization. In addition, the respiratory model enabled the modular insertion of interesting cell types, such as innate immune cells or multipotent stromal cells, showing a functional ability to release pertinent cytokines differentially. Furthermore this model responded imitating known events occurring during the infection by Non-typeable H. influenzae. Epithelial organoid models, mimicking the small intestine tract, were used for a different explorative analysis of tissue-toxicity. Further experiments led to detection of a cell population targeted by C. difficile Toxin A and suggested a role in the impairment of the epithelial homeostasis by the bacterial virulence machinery. The described cell-centered strategy can afford critical insights in the evaluation of the host defence and pathogenic mechanisms. The application of these two models may provide an informing step that more coherently defines relevant molecular interactions happening during the infection.

OPA1 isoforms and protein domains in the rescue of mitochondrial dysfunctions

Mutations in OPA1 gene have been identified in the majority of patients with Dominant Optic Atrophy (DOA), a blinding disease, and the syndromic form DOA-plus. OPA1 protein is a mitochondrial GTPase involved in various mitochondrial functions, present in humans in eight isoforms, resulting from alternative splicing and proteolytic processing. In this study we have investigated the specific role of each isoform through expression in OPA-/- MEFs, by evaluating their ability to improve the defective mitochondrial phenotypes. All isoforms were able to rescue the energetic efficiency, mitochondrial DNA (mtDNA) content and cristae integrity, but only the presence of both long and short forms could recover the mitochondrial morphology. In order to identify the OPA1 protein domains crucial for its functions, we selected and modified the isoform 1, shown to be one of the most efficient in preserving mitochondrial phenotype, to express three specific OPA1 variants, namely: one with a different N-terminus portion, one unable to generate short form owing to deletion of S1 cleavage site and one with a defective GTPase domain. We demonstrated that the simultaneous presence of the N- and C-terminus of OPA1 was essential for the mtDNA maintenance; a cleavable isoform generating s-forms was necessary to completely rescue the energetic competence and the presence of the C-terminus was sufficient to partially recover the cristae ultrastructure. Lastly, several pathogenic OPA1 mutations were inserted in MEF clones and the biochemical features investigated, to correlate the defective phenotypes with the clinical severity of patients. Our results clearly indicate that this cell model reflects very well the clinical characteristics of the patients, and therefore can be proposed as an useful tool to shed light on the pathomechanism underlying DOA.

Epigenetic role of N-Myc in Neuroblastoma

Childhood neuroblastoma is the most common solid tumour of infancy and highly refractory to therapy. One of the most powerful prognostic indicators for this disease is the N-Myc gene amplification, which occurs in approximately 25% of all neuroblastomas. N-Myc is a member of transcription factors belonging to a subclass of the larger group of proteins sharing Basic-Region/Helix–Loop–Helix/Leucin-Zipper (BR/HLH/LZ) motif. N-Myc oncoproteins may determine activation or repression of several genes thanks to different protein-protein interactions that may modulate its transcriptional regulatory ability and therefore its potential for oncogenicity. Chromatin modifications, including histone methylation, have a crucial role in transcription de-regulation of many cancer-related genes. Here, it was investigated whether N-Myc can functionally and/or physically interact with two different factors involved in methyl histone modification: WDR5 (core member of the MLL/Set1 methyltransferase complex) and the de- methylase LSD1. Co-IP assays have demonstrated the presence of both N-Myc-WDR5 and N-Myc-LSD1 complexes in two neuroblastoma cell lines. Human N-Myc amplified cell lines were used as a model system to investigate on transcription activation and/or repression mechanisms carried out by N-Myc-LSD1 and N-Myc-WDR5 protein complexes. qRT-PCR and immunoblot assays underlined the ability of both complexes to positively (N-Myc-WDR5) and negatively (N-Myc-LSD1) influence transcriptional regulation of crititical neuroblastoma N-Myc-related genes, MDM2, p21 and Clusterin. Ch-IP experiments have revealed the binding of the N-Myc complexes above mentioned to the gene promoters analysed. Finally, pharmacological treatment pointed to abolish N-Myc and LSD1 activity were performed to test cellular alterations, such as cell viability and cell cycle progression. Overall, the results presented in this work suggest that N-Myc can interact with two distinct histone methyl modifiers to positively and negatively affect gene transcription in neuroblastoma.

Study of the genes involved in Naphthenic acids (NAs) degradation by Rhodococcus spp.

Naphthenic acids (NAs) are an important group of organic pollutants mainly found in hydrocarbon deposits. Although these compounds are toxic, recalcitrant, and persistent in the environment, we are just learning the diversity of microbial communities involved in NAs- degradation and the mechanisms by which NAs are biodegraded. Studies have shown that naphthenic acids are susceptible to biodegradation, which decreases their concentration and reduces toxicity. Nevertheless, little is still known about their biodegradability. The present PhD Thesis’s work is aimed to study the biodegradation of simple model NAs using bacteria strains belonging to the Rhodococcus genus. In particular, Rh. sp. BCP1 and Rh. opacus R7 were able to utilize NAs such as cyclohexane carboxylic acid and cyclopentane carboxylic acid as the sole carbon and energy sources, even at concentrations up to 1000 mg/L. The presence of either substituents or longer carboxylic acid chains attached to the cyclohexane ring negatively affected the growth by pure bacterial cultures. Moreover, BCP1 and R7 cells incubated in the presence of CHCA or CPCA show a general increase of saturated and methyl-substituted fatty acids in their membrane, while the cis-mono-unsaturated ones decrease, as compared to glucose-grown cells. The observed lipid molecules modification during the growth in the presence of NAs is suggested as a possible mechanism to decrease the fluidity of the cell membrane to counteract NAs toxicity. In order to further evaluate this toxic effect on cell features, the morphological changes of BCP1 and R7 cells were also assessed through Transmission Electron Microscopy (TEM), revealing interesting ultrastructural changes. The induction of putative genes, and the construction of a random transposon mutagenesis library were also carried out to reveal the mechanisms by which these Rhodococcus strains can degrade toxic compounds such as NAs.