Molecular Characterization of Severe Acute Respiratory Syndrome Coronavirus 2 Isolates From Central Inner Sardinia.

BACKGROUND: The SARS-CoV-2 pandemic stimulated an outstanding global sequencing effort, which allowed to monitor viral circulation and evolution. Nuoro province (Sardinia, Italy), characterized by a relatively isolated geographical location and a low population density, was severely hit and displayed a high incidence of infection. METHODS: Amplicon approach Next Generation Sequencing and subsequent variant calling in 92 respiratory samples from SARS-CoV-2 infected patients involved in infection clusters from March 2020 to May 2021. RESULTS: Phylogenetic analysis displayed a coherent distribution of sequences in terms of lineage and temporal evolution of pandemic. Circulating lineage/clade characterization highlighted a growing diversity over time, with an increasingly growing number of mutations and variability of spike and nucleocapsid proteins, while viral RdRp appeared to be more conserved. A total of 384 different mutations were detected, of which 196 were missense and 147 synonymous ones. Mapping mutations along the viral genome showed an irregular distribution in key genes. S gene was the most mutated gene with missense and synonymous variants frequencies of 58.8 and 23.5%, respectively. Mutation rates were similar for the S and N genes with one mutation every ∼788 nucleotides and every ∼712 nucleotides, respectively. Nsp12 gene appeared to be more conserved, with one mutation every ∼1,270 nucleotides. The frequency of variant Y144F in the spike protein deviated from global values with higher prevalence of this mutation in the island. CONCLUSION: The analysis of the 92 viral genome highlighted evolution over time and identified which mutations are more widespread than others. The high number of sequences also permits the identification of subclusters that are characterized by subtle differences, not only in terms of lineage, which may be used to reconstruct transmission clusters. The disclosure of viral genetic diversity and timely identification of new variants is a useful tool to guide public health intervention measures.

Expression profiling of Mycobacterium tuberculosis H37Rv and Mycobacterium smegmatis in acid-nitrosative multi-stress displays defined regulatory networks.

Several studies regarding the transcriptome of Mycobacterium tuberculosis following the exposure to various in vitro simulated phagosomal stressors, have already tried to elucidate the bacterium behavior during the intracellular infection. An in vitro acid-nitrosative multi-stress was carried out for M. tuberculosis H37Rv and Mycobacterium smegmatis MC(2)155 in order to analyze by DNA-microarray the gene expression changes associated respectively to pathogenic and non-pathogenic mycobacterial species. During acid-nitrosative multi-stress both mycobacteria shift their transcriptome to allow the anaerobic respiratory state and energy pathways characteristic of starvation. M. tuberculosis counteracts the combined acid-nitrosative stress more efficiently than M. smegmatis as also shown by the up-regulation of glbN and hmp genes, that are specifically directed to NO detoxification. Moreover, the down-regulation of some virulence factors involved in phthiocerol dimycocerosates synthesis strengthens the hypothesis that these major virulence determinants may be attenuated by M. tuberculosis in the presence of reactive nitrogen species. In fact, it down-regulates other genes implicated in the synthesis of membrane structural lipids but in contrast to M. smegmatis, M. tuberculosis up-regulates many genes annotated for the synthesis of peptidoglycan. Results suggest a gene regulation of M. tuberculosis which reveals a distinctive expression pattern under stressful environment.

Unraveling the transcriptional regulatory networks associated to mycobacterial cell wall defective form induction by glycine and lysozyme treatment.

It is known that a combined glycine/lysozyme treatment is able to induce in vitro the mycobacterial conversion from the bacillary to the cell wall defective forms. These forms also naturally occur in vivo as a response to various antimicrobial factors such as lysozyme released by phagocytic cells. Although they have been successfully isolated from patients with several chronic diseases, their role in pathogenesis is still unknown, mainly due to the difficulties in handling the in vivo isolated variants. Moreover, nothing is known about the transcriptional peculiarities that may exist in comparison to the vegetative phase. Hence, in this study, we simulated in vitro the induction of the mycobacterial cell wall defective state by using a glycine and lysozyme-based treatment in order to identify the gene expression profiles of both pathogenic and non-pathogenic mycobacteria. DNA-microarray results showed that in contrast to the non-pathogenic Mycobacterium smegmatis species, glycine and lysozyme treated forms of Mycobacterium tuberculosis and Mycobacterium avium subspecies paratuberculosis regulated a repertoire of genes usually expressed in vivo during adaptation and persistence within host environments. Results suggest that the cell wall defective state may represent an important stage in the life-cycle of pathogenic mycobacteria that potentially coordinates persistence.

Gene expression profiling of Mycobacterium avium subsp. paratuberculosis in simulated multi-stress conditions and within THP-1 cells reveals a new kind of interactive intramacrophage behaviour.

BACKGROUND: Recent studies have identified in Mycobacterium avium subsp. paratuberculosis (MAP), already known as a pathogen in ruminants, a potential zoonotic agent of some autoimmune diseases in humans. Therefore, considering the possible risk for public health, it is necessary a thorough understanding of MAP's gene expression during infection of human host as well as the identification of its immunogenic and/or virulence factors for the development of appropriate diagnostic and therapeutic tools. RESULTS: In order to characterize MAP's transcriptome during macrophage infection, we analyzed for the first time the whole gene expression of a human derived strain of MAP in simulated intraphagosomal conditions and after intracellular infection of the human macrophage cell line THP-1 by using the DNA-microarray technology. Results showed that MAP shifts its transcriptome to an adaptive metabolism for an anoxic environment and nutrient starvation. It up-regulates several response factors to oxidative stress or intracellular conditions and allows, in terms of transcription, a passive surface peptidoglycan spoliation within the macrophage along with an intensification of the anabolic activity for lipidic membrane structures. CONCLUSIONS: These results indicate a possible interactive system between MAP and its host cell based on the internal mimicry unlike other intracellular pathogens, bringing new hypothesis in the virulence and pathogenicity of MAP and its importance in human health.

Detection of pathogenic mycobacteria based on functionalized quantum dots coupled with immunomagnetic separation.

Mycobacteria have always proven difficult to identify due to their low growth rate and fastidious nature. Therefore molecular biology and more recently nanotechnology, have been exploited from early on for the detection of these pathogens. Here we present the first stage of development of an assay incorporating cadmium selenide quantum dots (QDs) for the detection of mycobacterial surface antigens. The principle of the assay is the separation of bacterial cells using magnetic beads coupled with genus-specific polyclonal antibodies and monoclonal antibodies for heparin-binding hemagglutinin. These complexes are then tagged with anti-mouse biotinylated antibody and finally streptavidin-conjugated QDs which leads to the detection of a fluorescent signal. For the evaluation of performance, the method under study was applied on Mycobacterium bovis BCG and Mycobacterium tuberculosis (positive controls), as well as E. coli and Salmonella spp. that constituted the negative controls. The direct observation of the latter category of samples did not reveal fluorescence as opposed to the mycobacteria mentioned above. The minimum detection limit of the assay was defined to 10(4) bacteria/ml, which could be further decreased by a 1 log when fluorescence was measured with a spectrofluorometer. The method described here can be easily adjusted for any other protein target of either the pathogen or the host, and once fully developed it will be directly applicable on clinical samples.

MAP3738c and MptD are specific tags of Mycobacterium avium subsp. paratuberculosis infection in type I diabetes mellitus.

Mycobacterium avium subsp. paratuberculosis (Map) is the causative agent of Johne's disease, a chronic inflammation of ruminants' intestine. Recent studies have linked Map to type I Diabetes mellitus (T1DM). We searched the presence of antibodies against two specific proteins of Map (MptD and MAP3738c) in sera of patients affected by T1DM and type II Diabetes mellitus (T2DM). MptD protein (MAP3733c) has been recognized as a Map virulent factor whereas MAP3738c has not yet been studied. Both proteins are encoded by genes belonging to a Map specific pathogenicity island. Forty three T1DM patients' sera, 56 T2DM patients' sera and 48 healthy subjects' sera were screened by ELISA to evaluate the immunoresponse against MptD or MAP3738c recombinant proteins. Results showed a positive response to both proteins in T1DM patients whereas no difference with controls was found for T2DM patients. Results suggest a potential relation between T1DM and the bacterial infection.

Phages specific for mycobacterial lipoarabinomannan help serodiagnosis of tuberculosis.

This study evaluated the possibility to use six phages specific to the Mycobacterium tuberculosis lipoarabinomannan (LAM) as tools for tubercular serodiagnosis. We analysed sera samples from 30 subjects with active tuberculosis (TB+), 30 with latent tubercular infection (LTBI) and 60 healthy subjects as controls (K). Our data indicated a good antibody response of the TB+ and LTBI patients against the phage Ri(7)17; the optical density (OD) values obtained from sera patients was statistically significant when compared to the control samples. Our results confirm that phage display technology might be useful to develop new tools for diagnosis of tuberculosis.

Linking chronic infection and autoimmune diseases: Mycobacterium avium subspecies paratuberculosis, SLC11A1 polymorphisms and type-1 diabetes mellitus.

BACKGROUND: The etiology of type 1 diabetes mellitus (T1DM) is still unknown; numerous studies are performed to unravel the environmental factors involved in triggering the disease. SLC11A1 is a membrane transporter that is expressed in late endosomes of antigen presenting cells involved in the immunopathogenic events leading to T1DM. Mycobacterium avium subsp. paratuberculosis (MAP) has been reported to be a possible trigger in the development of T1DM. METHODOLOGY/PRINCIPAL FINDINGS: Fifty nine T1DM patients and 79 healthy controls were genotyped for 9 polymorphisms of SLC11A1 gene, and screened for the presence of MAP by PCR. Differences in genotype frequency were evaluated for both T1DM patients and controls. We found a polymorphism in the SLC11A1 gene (274C/T) associated to type 1 diabetic patients and not to controls. The presence of MAP DNA was also significantly associated with T1DM patients and not with controls. CONCLUSIONS/SIGNIFICANCE: The 274C/T SCL11A1 polymorphism was found to be associated with T1DM as well as the presence of MAP DNA in blood. Since MAP persists within macrophages and it is also processed by dendritic cells, further studies are necessary to evaluate if mutant forms of SLC11A1 alter the processing or presentation of MAP antigens triggering thereby an autoimmune response in T1DM patients.

Specific immunoassays confirm association of Mycobacterium avium Subsp. paratuberculosis with type-1 but not type-2 diabetes mellitus.

BACKGROUND: Mycobacterium avium subspecies paratuberculosis (MAP) is a versatile pathogen with a broad host range. Its association with type-1 diabetes mellitus (T1DM) has been recently proposed. Rapid identification of infectious agents such as MAP in diabetic patients at the level of clinics might be helpful in deciphering the role of chronic bacterial infection in the development of autoimmune diseases such as T1DM. METHODOLOGY/PRINCIPAL FINDINGS: We describe use of an ELISA method to identify live circulating MAP through the detection of a cell envelope protein, MptD by a specific M13 phage--fMptD. We also used another ELISA format to detect immune response to MptD peptide. Both the methods were tested with blood plasma obtained from T1DM, type-2 diabetes (T2DM) patients and non-diabetic controls. Our results demonstrate MptD and fMptD ELISA assays to be accurate and sensitive to detect MAP bacilli in a large fraction (47.3%) of T1DM patients as compared to non-diabetic controls (12.6%) and those with confirmed T2DM (7.7%). Comparative analysis of ELISA assays performed here with 3 other MAP antigen preparations, namely HbHA, Gsd and whole cell MAP lysates confirmed comparable sensitivity of the MptD peptide and the fMptD based ELISA assays. Moreover, we were successful in demonstrating positive bacterial culture in two of the clinical specimen derived from T1DM patients. CONCLUSIONS AND SIGNIFICANCE: The MptD peptide/fMptD based ELISA or similar tests could be suggested as rapid and specific field level diagnostic tests for the identification of MAP in diabetic patients and for finding the explanations towards the occurrence of type-1 or type-2 diabetes in the light of an active infectious trigger.

Mycobacterium avium subspecies paratuberculosis is not associated with type-2 diabetes mellitus.

BACKGROUND: The role of pathogenic mycobacteria in diabetes has been a focus of speculation since a decade without any meaningful insights into the mechanism of diabetes causation vis a vis mycobacterial factors. Two of our studies based on PCR identification of mycobacterial DNA and detection of antibodies specific to the recombinant antigens and whole cell lysates of the Mycobacterium avium subsp. paratuberculosis (MAP) shown a clear association of MAP with the presence of type 1 diabetes mellitus (T1DM). METHODS: In this study, we sought to investigate if or not type 2 diabetes (T2DM) patients harbour humoral responses to MAP. Using three different MAP antigen preparations, humoral antibody profiles were estimated for 57 T2DM patients and 57 healthy controls. Statistical analysis was performed with the Chi-square test with Yates' corrections. RESULTS: We observed insignificant levels of humoral antibodies against recombinant heparin binding haemagglutinin (HbHA), glycosyl transferase (Gsd) and MAP whole cell lysate in the blood of subjects with T2DM as compared to healthy controls. CONCLUSION: We found no obvious association of MAP with the incidence of T2DM in Sardinian patients.

Humoral immune responses of type 1 diabetes patients to Mycobacterium avium subsp. paratuberculosis lend support to the infectious trigger hypothesis.

Mycobacterium avium subsp. paratuberculosis is a zoonotic pathogen whose association with Crohn's disease in humans is under scrutiny. The objective of this work was to investigate its association with other chronic diseases such as type 1 diabetes mellitus (T1DM), where the involvement of a persistent pathogen such as M. avium subsp. paratuberculosis could be the trigger. For this purpose, 59 diabetic patients and 59 healthy controls were investigated for the presence of antibodies against two recombinant proteins of M. avium subsp. paratuberculosis and the whole-cell lysate. Extremely significant humoral immune responses to recombinant heparin binding hemagglutinin and glycosyl transferase proteins and the whole-cell lysates of M. avium subsp. paratuberculosis bacilli were observed in T1DM patients and compared to those of healthy controls. Finding evidence of M. avium subsp. paratuberculosis involvement in T1DM is perhaps a novel finding that might serve as a foundation stone in establishing an infectious etiology for T1DM.

Antigenic profiles of recombinant proteins from Mycobacterium avium subsp. paratuberculosis in sheep with Johne's disease.

Methods to improve the ELISA test to detect Mycobacterium avium subsp. paratuberculosis have been explored over several years. Previously, selected recombinant proteins of M. avium subspecies paratuberculosis were found to be immunogenic in cattle with Johne's disease. In the present study, antibody responses of infected and healthy sheep were evaluated using 18 purified recombinant proteins in an ELISA-based format for the serodiagnosis of ovine paratuberculosis. These selected recombinant proteins represent heat shock proteins, hypothetical proteins and cell surface proteins of M. avium subsp. paratuberculosis. Whereas, Map0862 (a gene uniquely present in M. avium subspecies paratuberculosis) and Map3786 encoded protein solicited the strongest antibody response in infected sheep. The protein encoded by Map2116c showed the weakest antibody response among the animals tested. Although none of the recombinant proteins detected all 11 infected sheep singly, antibodies to Map0862 were detected in 9 of 11 (81%) infected sheep. Furthermore, ovine responses to these selected antigens were assessed temporally over the course of 1 year during which we found a spiking effect rather than an incremental increase of antibody reactivity. This study evaluated multiple M. avium subsp. paratuberculosis recombinant proteins in an ELISA-based format for sheep.

Effects of crp deletion in Salmonella enterica serotype Gallinarum.

BACKGROUND: Salmonella enterica serotype Gallinarum (S. Gallinarum) remains an important pathogen of poultry, especially in developing countries. There is a need to develop effective and safe vaccines. In the current study, the effect of crp deletion was investigated with respect to virulence and biochemical properties and the possible use of a deletion mutant as vaccine candidate was preliminarily tested. METHODS: Mutants were constructed in S. Gallinarum by P22 transduction from Salmonella Typhimurium (S. Typhimurium) with deletion of the crp gene. The effect was characterized by measuring biochemical properties and by testing of invasion in a chicken loop model and by challenge of six-day-old chickens. Further, birds were immunized with the deleted strain and challenged with the wild type isolate. RESULTS: The crp deletions caused complete attenuation of S. Gallinarum. This was shown by ileal loop experiments not to be due to significantly reduced invasion. Strains with such deletions may have vaccine potential, since oral inoculatoin with S. Gallinarum Deltacrp completely protected against challenge with the same dose of wild type S. Gallinarum ten days post immunization. Interestingly, the mutations did not cause the same biochemical and growth changes to the two biotypes of S. Gallinarum. All biochemical effects but not virulence could be complemented by providing an intact crp-gene from S. Typhimurium on the plasmid pSD110. CONCLUSION: Transduction of a Tn10 disrupted crp gene from S. Typhimurium caused attenuation in S. Gallinarum and mutated strains are possible candidates for live vaccines against fowl typhoid.

sigma28-dependent transcription in Salmonella enterica is independent of flagellar shearing.

The FlgM anti-sigma28 factor is secreted in response to flagellar hook-basal body completion to allow sigma28-dependent transcription of genes needed late in flagellar assembly, such as the flagellin structural gene, fliC. A long-standing hypothesis was that one role of FlgM secretion was to allow rapid expression of flagellin in response to shearing. We tested this hypothesis by following FlgM secretion and fliC transcription in response to flagellar shearing. Experiments showed that the level of FlgM inside the cell was unchanged after shearing whereas the extracellular FlgM levels increased in the growth medium as time passed. Identical results were obtained with cells that were not exposed to shear forces: internal FlgM levels remained constant while external FlgM levels rose with time at rates similar to those for the sheared culture. Consistent with this find, FlgM/sigma28-dependent class 3 gene expression was unaffected by flagellar shearing but was affected by the growth phase of the cell. Regardless of exposure to shear forces, flagellar class 3 transcription rose sharply and then declined. These results demonstrate that flagellar regrowth following shearing is independent of FlgM secretion.

Translation inhibition of the Salmonella fliC gene by the fliC 5' untranslated region, fliC coding sequences, and FlgM.

The 5'-untranslated region (5'UTR) of the fliC flagellin gene of Salmonella contains sequences critical for efficient fliC mRNA translation coupled to assembly. In a previous study we used targeted mutagenesis of the 5' end of the fliC gene to isolate single base changes defective in fliC gene translation. This identified a predicted stem-loop structure, SL2, as an effector of normal fliC mRNA translation. A single base change (-38C:U) in the fliC 5'UTR resulted in a mutant that is defective in fliC mRNA translation and was chosen for this study. Motile (Mot+) revertants of the -38C:T mutant were isolated and characterized, yielding several unexpected results. Second-site suppressors that restored fliC translation and motility included mutations that disrupt a RNA duplex stem formed between RNA sequences in the fliC 5'UTR SL2 region (including a precise deletion of SL2) and bases early within the fliC-coding region. A stop codon mutation at position 80 of flgM also suppressed the -38C:T motility defect, while flgM mutants defective in anti-sigma28 activity had no effect on fliC translation. One remarkable mutation in the fliC 5'UTR (-15G:A) results in a translation defect by itself but, in combination with the -38C:U mutation, restores normal translation. These results suggests signals intrinsic to the fliC mRNA that have both positive and negative effects on fliC translation involving both RNA structure and interacting proteins.