A clickable melphalan for monitoring DNA interstrand crosslink accumulation and detecting ICL repair defects in Fanconi anemia patient cells.

Fanconi anemia (FA) is a genetic disorder associated with developmental defects, bone marrow failure and cancer. The FA pathway is crucial for the repair of DNA interstrand crosslinks (ICLs). In this study, we have developed and characterized a new tool to investigate ICL repair: a clickable version of the crosslinking agent melphalan which we name click-melphalan. Our results demonstrate that click-melphalan is as effective as its unmodified counterpart in generating ICLs and associated toxicity. The lesions induced by click-melphalan can be detected in cells by post-labelling with a fluorescent reporter and quantified using flow cytometry. Since click-melphalan induces both ICLs and monoadducts, we generated click-mono-melphalan, which only induces monoadducts, in order to distinguish between the two types of DNA repair. By using both molecules, we show that FANCD2 knock-out cells are deficient in removing click-melphalan-induced lesions. We also found that these cells display a delay in repairing click-mono-melphalan-induced monoadducts. Our data further revealed that the presence of unrepaired ICLs inhibits monoadduct repair. Finally, our study demonstrates that these clickable molecules can differentiate intrinsic DNA repair deficiencies in primary FA patient cells from those in primary xeroderma pigmentosum patient cells. As such, these molecules may have potential for developing diagnostic tests.

B-cell non-Hodgkin lymphoma linked to Coxiella burnetii.

Bacteria can induce human lymphomas, whereas lymphoproliferative disorders have been described in patients with Q fever. We observed a lymphoma in a patient with Q fever that prompted us to investigate the association between the 2 diseases. We screened 1468 consecutive patients of the 2004 to 2014 French National Referral Center for Q fever database. The standardized incidence ratios (SIRs) of diffuse large B-cell lymphoma (DLBCL) and follicular lymphoma (FL) were calculated comparatively to the 2012 Francim Registry. The presence of Coxiella burnetii was tested using immunofluorescence and fluorescence in situ hybridization using a specific 16S ribosomal RNA probe and genomic DNA probe. Seven patients (0.48%) presented mature B-cell lymphoma consisting of 6 DLBCL and 1 FL. An excess risk of DLBCL and FL was found in Q fever patients compared with the general population (SIR [95% confidence interval], 25.4 [11.4-56.4] and 6.7 [0.9-47.9], respectively). C burnetii was detected in CD68(+) macrophages within both lymphoma and lymphadenitis tissues but localization in CD123(+) plasmacytoid dendritic cells (pDCs) was found only in lymphoma tissues. Q fever patients with persistent focalized infection were found more at risk of lymphoma (hazard ratio, 9.35 [1.10-79.4]). Interleukin-10 (IL10) overproduction (P = .0003) was found in patients developing lymphoma. These results suggest that C burnetii should be added to the list of bacteria that promote human B-cell non-Hodgkin lymphoma, possibly by the infection of pDCs and IL10 overproduction. Screening for early lymphoma diagnosis should be considered in the management of patients with Q fever, especially those with persistent focalized infections.

Case report: Coxiella burnetii vascular infection and lymphoma in the Netherlands.

OBJECTIVES AND DESIGN: Non-Hodgkin lymphoma has been linked to infection with Coxiella burnetii, potentially through overproduction of IL-10 during infection with C. burnetii. MATERIALS AND METHODS: Description of a case report. RESULTS: We describe a patient with retroperitoneal non-Hodgkin lymphoma and vascular infection with C. burnetii. Immunofluorescence staining and fluorescence in situ hybridization targeting specific C. burnetii 16S rRNA were performed on the retroperitoneal lymphoma tissue sample obtained at diagnosis of NHL. Both were strongly positive for the presence of C. burnetii. CONCLUSIONS: This case provokes questions regarding a potential association between C. burnetii and NHL, and underlines the importance of further exploration of this association.

Transmission potential of Rickettsia felis infection by Anopheles gambiae mosquitoes.

A growing number of recent reports have implicated Rickettsia felis as a human pathogen, paralleling the increasing detection of R. felis in arthropod hosts across the globe, primarily in fleas. Here Anopheles gambiae mosquitoes, the primary malarial vectors in sub-Saharan Africa, were fed with either blood meal infected with R. felis or infected cellular media administered in membrane feeding systems. In addition, a group of mosquitoes was fed on R. felis-infected BALB/c mice. The acquisition and persistence of R. felis in mosquitoes was demonstrated by quantitative PCR detection of the bacteria up to day 15 postinfection. R. felis was detected in mosquito feces up to day 14. Furthermore, R. felis was visualized by immunofluorescence in salivary glands, in and around the gut, and in the ovaries, although no vertical transmission was observed. R. felis was also found in the cotton used for sucrose feeding after the mosquitoes were fed infected blood. Natural bites from R. felis-infected An. gambiae were able to cause transient rickettsemias in mice, indicating that this mosquito species has the potential to be a vector of R. felis infection. This is particularly important given the recent report of high prevalence of R. felis infection in patients with "fever of unknown origin" in malaria-endemic areas.

Mouse Model of Coxiella burnetii Aerosolization.

Coxiella burnetii is mainly transmitted by aerosols and is responsible for multiple-organ lesions. Animal models have shown C. burnetii pathogenicity, but long-term outcomes still need to be clarified. We used a whole-body aerosol inhalation exposure system to mimic the natural route of infection in immunocompetent (BALB/c) and severe combined immunodeficient (SCID) mice. After an initial lung inoculum of 10(4) C. burnetii cells/lung, the outcome, serological response, hematological disorders, and deep organ lesions were described up to 3 months postinfection. C. burnetii-specific PCR, anti-C. burnetii immunohistochemistry, and fluorescent in situ hybridization (FISH) targeting C. burnetii-specific 16S rRNA completed the detection of the bacterium in the tissues. In BALB/c mice, a thrombocytopenia and lymphopenia were first observed, prior to evidence of C. burnetii replication. In all SCID mouse organs, DNA copies increased to higher levels over time than in BALB/c ones. Clinical signs of discomfort appeared in SCID mice, so follow-up had to be shortened to 2 months in this group. At this stage, all animals presented bone, cervical, and heart lesions. The presence of C. burnetii could be attested in situ for all organs sampled using immunohistochemistry and FISH. This mouse model described C. burnetii Nine Mile strain spread using aerosolization in a way that corroborates the pathogenicity of Q fever described in humans and completes previously published data in mouse models. C. burnetii infection occurring after aerosolization in mice thus seems to be a useful tool to compare the pathogenicity of different strains of C. burnetii.

Crystal structure of the VgrG1 actin cross-linking domain of the Vibrio cholerae type VI secretion system.

Vibrio cholerae is the cause of the diarrheal disease cholera. V. cholerae produces RtxA, a large toxin of the MARTX family, which is targeted to the host cell cytosol, where its actin cross-linking domain (ACD) cross-links G-actin, leading to F-actin depolymerization, cytoskeleton rearrangements, and cell rounding. These effects on the cytoskeleton prevent phagocytosis and bacterial engulfment by macrophages, thus preventing V. cholerae clearance from the gut. The V. cholerae Type VI secretion-associated VgrG1 protein also contains a C-terminal ACD, which shares 61% identity with MARTX ACD and has been shown to covalently cross-link G-actin. Here, we purified the VgrG1 C-terminal domain and determined its crystal structure. The VgrG1 ACD exhibits a V-shaped three-dimensional structure, formed of 12 ß-strands and nine α-helices. Its active site comprises five residues that are conserved in MARTX ACD toxin, within a conserved area of ∼10 Å radius. We showed that less than 100 ACD molecules are sufficient to depolymerize the actin filaments of a fibroblast cell in vivo. Mutagenesis studies confirmed that Glu-16 is critical for the F-actin depolymerization function. Co-crystals with divalent cations and ATP reveal the molecular mechanism of the MARTX/VgrG toxins and offer perspectives for their possible inhibition.

Impaired virulence and fitness of a colistin-resistant clinical isolate of Acinetobacter baumannii in a rat model of pneumonia.

We compared the fitness and lung pathogenicity of two isogenic clinical isolates of Acinetobacter baumannii, one resistant (ABCR) and the other susceptible (ABCS) to colistin. In vitro, ABCR exhibited slower growth kinetics than ABCS. In a rat model of pneumonia, ABCR was associated with less pronounced signs of infection (lung bacterial count, systemic dissemination, and lung damage) and a better outcome (ABCR and ABCS mortality rates, 20 and 50%, respectively [P = 0.03]).

UFMylation of MRE11 is essential for telomere length maintenance and hematopoietic stem cell survival.

Ubiquitin-fold modifier 1 (UFM1) is involved in neural and erythroid development, yet its biological roles in these processes are unknown. Here, we generated zebrafish models deficient in Ufm1 and Ufl1 that exhibited telomere shortening associated with developmental delay, impaired hematopoiesis and premature aging. We further report that HeLa cells lacking UFL1 have instability of telomeres replicated by leading-strand synthesis. We uncover that MRE11 UFMylation is necessary for the recruitment of the phosphatase PP1-α leading to dephosphorylation of NBS1. In the absence of UFMylation, NBS1 remains phosphorylated, thereby reducing MRN recruitment to telomeres. The absence of MRN at telomeres favors the formation of the TRF2-Apollo/SNM1 complex consistent with the loss of leading telomeres. These results suggest that MRE11-UFMylation may serve as module to recruit PP1-α. Last, zebrafish expressing Mre11 that cannot be UFMylated phenocopy Ufm1-deficient zebrafish, demonstrating that UFMylation of MRE11 is a previously undescribed evolutionarily conserved mechanisms regulating telomere length.

HIV-2 Protease resistance defined in yeast cells.

BACKGROUND: Inhibitors of the HIV-1 Protease currently used in therapeutic protocols, have been found to inhibit, although at higher concentrations, the HIV-2 encoded enzyme homologue. Similar to observations in HIV-1 infected individuals, therapeutic failure has also been observed for some patients infected with HIV-2 as a consequence of the emergence of viral strains resistant to the anti-retroviral molecules. In order to be able to define the specific mutations in the Protease that confer loss of susceptibility to Protease Inhibitors, we set up an experimental model system based in the expression of the viral protein in yeast. RESULTS: Our results show that the HIV-2 Protease activity kills the yeast cell, and this process can be abolished by inhibiting the viral enzyme activity. Since this inhibition is dose dependent, IC50 values can be assessed for each anti-retroviral molecule tested. We then defined the susceptibility of HIV-2 Proteases to Protease Inhibitors by comparing the IC50 values of Proteases from 7 infected individuals to those of a sensitive wild type laboratory adapted strain. CONCLUSION: This functional assay allowed us to show for the first time that the L90M substitution, present in a primary HIV-2 isolate, modifies the HIV-2 Protease susceptibility to Saquinavir but not Lopinavir. Developing a strategy based on the proposed yeast expressing system will contribute to define amino acid substitutions conferring HIV-2 Protease resistance.

Deglycosylation of Tropheryma whipplei biofilm and discrepancies between diagnostic results during Whipple's disease progression.

Whipple's disease is a systemic infectious disease associated with the bacterium Tropheryma whipplei. Numerous reports have presented puzzling discrepancies between diagnosis methods. We addressed this confusion using fluorescent in situ hybridization and immunofluorescence assays to evaluate 34 duodenal biopsies and 1 lymph node biopsy from Whipple's patients. We showed the presence of bacteria in both CK20(+) epithelial cells and CD68(+) macrophages. Bacteria are found embedded in a biofilm hindering the detection of T. whipplei. Only after treatment of biopsies by glycosidases, co-localization of T. whipplei RNA/DNA with bacterial proteins was restored. Moreover, using 13 bronchoalveolar lavages and 7 duodenal biopsies, we found that hydrolysis of the biofilm weakened the bacteria, facilitated bacterial DNA extraction and improved the sensitivity of qPCR detection by up to 1000x opening new perspectives for diagnostic and scientific approaches.

Glycans affect DNA extraction and induce substantial differences in gut metagenomic studies.

Exopolysaccharides produced by bacterial species and present in feces are extremely inhibitory to DNA restriction and can cause discrepancies in metagenomic studies. We determined the effects of different DNA extraction methods on the apparent composition of the gut microbiota using Illumina MiSeq deep sequencing technology. DNA was extracted from the stool from an obese female using 10 different methods and the choice of DNA extraction method affected the proportional abundance at the phylum level, species richness (Chao index, 227 to 2,714) and diversity (non parametric Shannon, 1.37 to 4.4). Moreover DNA was extracted from stools obtained from 83 different individuals by the fastest extraction assay and by an extraction assay that degradated exopolysaccharides. The fastest extraction method was able to detect 68% to 100% genera and 42% to 95% species whereas the glycan degradation extraction method was able to detect 56% to 93% genera and 25% to 87% species. To allow a good liberation of DNA from exopolysaccharides commonly presented in stools, we recommend the mechanical lysis of stools plus glycan degradation, used here for the first time. Caution must be taken in the interpretation of current metagenomic studies, as the efficiency of DNA extraction varies widely among stool samples.

Restricted diversity of dental calculus methanogens over five centuries, France.

Methanogens are acknowledged archaeal members of modern dental calculus microbiota and dental pathogen complexes. Their repertoire in ancient dental calculus is poorly known. We therefore investigated archaea in one hundred dental calculus specimens collected from individuals recovered from six archaeological sites in France dated from the 14(th) to 19(th) centuries AD. Dental calculus was demonstrated by macroscopic and cone-beam observations. In 56 calculus specimens free of PCR inhibition, PCR sequencing identified Candidatus Methanobrevibacter sp. N13 in 44.6%, Methanobrevibacter oralis in 19.6%, a new Methanomassiliicoccus luminyensis-like methanogen in 12.5%, a Candidatus Nitrososphaera evergladensis-like in one and Methanoculleus bourgensis in one specimen, respectively. One Candidatus Methanobrevibacter sp. N13 dental calculus was further documented by fluorescent in situ hybridization. The prevalence of dental calculus M. oralis was significantly lower in past populations than in modern populations (P = 0.03, Chi-square test). This investigation revealed a previously unknown repertoire of archaea found in the oral cavity of past French populations as reflected in preserved dental calculus.

Marseillevirus in lymphoma: a giant in the lymph node.

The family Marseilleviridae is a new clade of giant viruses whose original member, marseillevirus, was described in 2009. These viruses were isolated using Acanthamoeba spp primarily from the environment. Subsequently, a close relative of marseillevirus was isolated from the faeces of a healthy young man, and others were detected in blood samples of blood donors and recipients and in a child with lymph node adenitis. In this Grand Round we describe the detection of marseillevirus by PCR, fluorescence in-situ hybridisation, direct immunofluorescence, and immunohistochemistry in the lymph node of a 30-year-old woman diagnosed with Hodgkin's lymphoma, together with IgG antibodies to marseillevirus. A link with viruses and bacteria has been reported for many lymphomas. We review the literature describing these associations, the criteria used to consider a causal association, and the underlying mechanisms of lymphomagenesis. Our observations suggest that consideration should be given to marseillevirus infections as an additional viral cause or consequence of Hodgkin's lymphoma, and that this hypothesis should be tested further.

Involvement of a small GTP binding protein in HIV-1 release.

BACKGROUND: There is evidence suggesting that actin binding to HIV-1 encoded proteins, or even actin dynamics themselves, might play a key role in virus budding and/or release from the infected cell. A crucial step in the reorganisation of the actin cytoskeleton is the engagement of various different GTP binding proteins. We have thus studied the involvement of GTP-binding proteins in the final steps of the HIV-1 viral replication cycle. RESULTS: Our results demonstrate that virus production is abolished when cellular GTP binding proteins involved in actin polymerisation are inhibited with specific toxins. CONCLUSION: We propose a new HIV budding working model whereby Gag interactions with pre-existing endosomal cellular tracks as well as with a yet non identified element of the actin polymerisation pathway are required in order to allow HIV-1 to be released from the infected cell.

Identification of flea species using MALDI-TOF/MS.

In the present study, a molecular proteomics (MALDI-TOF/MS) approach was used as a tool for identifying flea vectors. We measured the MS spectra from 38 flea specimens of 5 species including Ctenocephalides felis, Ctenocephalides canis, Archaeopsylla erinacei, Xenopsylla cheopis and Stenoponia tripectinata. A blind test performed with 24 specimens from species included in a library spectral database confirmed that MALDI-TOF/MS is an effective tool for discriminating flea species. Although fresh and 70% ethanol-conserved samples subjected to MALDI-TOF/MS in blind tests were correctly classified, only MS spectra of quality from fresh specimens were sufficient for accurate and significant identification. A cluster analysis highlighted that the MALDI Biotyper can be used for studying the phylogeny of fleas.

Connection of toxin-antitoxin modules to inoculation eschar and arthropod vertical transmission in Rickettsiales.

The biological role of toxin-antitoxin systems (TAS) in pathogenicity and cell addiction of Rickettsia was recently reported. We realized a comparative genomic analysis onto 33 rickettsial genomes and correlated the presence of TAS encoding genes with vertical transmission (VT) in arthropod hosts, the presence of inoculation eschar in humans and experimental animals, and the mortality in humans. There is a significant statistical link between TAS and the presence of an eschar (p≤0.0001). The presence of TAS is also significantly inversely correlated with mortality. The toxic effect of TAS may increase the local reaction, thus inhibiting the spread of rickettsiae associated with fatal outcome of the disease. The TAS were also linked to VT (p≤0.0001). Together with our previous findings we speculate that this is the first addiction system evidenced in intracellular bacteria. Thus, the TAS, as selfish genetic elements, might be essential to the evolutionary strategy of intracellular bacteria.

High-level colonisation of the human gut by Verrucomicrobia following broad-spectrum antibiotic treatment.

The gut microbiota is mainly composed of the phyla Firmicutes, Bacteroidetes, Actinobacteria and Proteobacteria; the Verrucomicrobia phylum is occasionally observed. Antibiotics can change the bacterial diversity of the gut, with limited changes in the proportions of phyla. In this study, the gut repertoire of two patients who received a broad-spectrum antibiotic regimen was studied. As part of a large gut microbiota study, two stool samples were analysed: one sample was collected after broad-spectrum antibiotic therapy in a patient with Coxiella burnetii vascular infection (Patient A); and the other sample was collected from a patient admitted to the Intensive Care Unit (Patient B). Samples were subjected to Gram staining, electron microscopy, 16S rRNA V6 amplicon pyrosequencing and fluorescence in situ hybridisation (FISH). In parallel, the antibiotic susceptibility of Akkermansia muciniphila Muc(T) strain was studied and this strain was observed by electron microscopy. Pyrosequencing revealed that a large proportion of the sequences were associated with Verrucomicrobia (proportions of 44.9% and 84.6% for Patients A and B, respectively). All of the phylotypes were represented by a single species (A. muciniphila), and neither patient presented significant gastrointestinal disorders. Electron microscopy and FISH with specific Verrucomicrobia probes confirmed the presence of the bacterium. The Muc(T) strain was susceptible to imipenem and doxycycline but resistant to vancomycin and metronidazole. Dramatic colonisation of the human gut microbiota by the Verrucomicrobia phylum following a broad-spectrum antibiotic regimen occurred without significant gastrointestinal manifestations, suggesting that influenced by external factors such as antibiotics, the gut repertoire remains partially unknown.

Evaluation of the diagnostic value of fluorescent in situ hybridization in a rat model of bacterial pneumonia.

In severe nosocomial pneumonia, the pathogenic responsibility of bacteria isolated from airways is far from certain, and a lung biopsy is sometimes performed. However, detection and identification of pathogens are frequently unachieved. Here, we developed a protocol for direct visualization of bacteria within the lung tissue using fluorescent in situ hybridization (FISH) in a rat model of Acinetobacter baumannii pneumonia. The reference positive diagnosis of bacterial pneumonia was the presence of pathological signs of pneumonia associated with the proof of bacteria or bacterial PCR products into the parenchyma. By analysis of 122 sets of slices from 26 rats and using the eubacterial probe EUB-338, our results show that FISH reached a sensitivity and a diagnostic accuracy higher than that of optic microscopy (sensitivity: 96% versus 55.4% and diagnostic accuracy: 96.7% versus 66.4%), whereas both approaches had 100% specificity. FISH could be useful especially on negative biopsies from patients with suspected infectious pneumonia.

Effect of rickettsial toxin VapC on its eukaryotic host.

Rickettsia are intracellular bacteria typically associated with arthropods that can be transmitted to humans by infected vectors. Rickettsia spp. can cause mild to severe human disease with a possible protection effect of corticosteroids when antibiotic treatments are initiated. We identified laterally transferred toxin-antitoxin (TA) genetic elements, including vapB/C, in several Rickettsia genomes and showed that they are functional in bacteria and eukaryotic cells. We also generated a plaque assay to monitor the formation of lytic plaques over time and demonstrated that chloramphenicol accelerates host cell lysis of vapB/C-containing Rickettsia. Whole-genome expression, TUNEL and FISH assays on the infected cells following exposure to the antibiotic revealed early apoptosis of host cells, which was linked to over-transcription of bacterial vapB/C operons and subsequent cytoplasmic VapC toxin release. VapC that is expressed in Escherichia coli and Saccharomyces cerevisiae or microinjected into mammalian cells is toxic through RNase activity and is prevented by dexamethasone. This study provides the first biological evidence that toxin-antitoxin elements act as pathogenic factors in bacterial host cells, confirming comparative genomic evidence of their role in bacterial pathogenicity. Our results suggest that early mortality following antibiotic treatment of some bacterial infections can be prevented by administration of dexamethasone.