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Thienamycin, the first representative of carbapenem antibiotics was discovered in the mid-1970s from soil microorganism, Streptomyces cattleya, during the race to discover inhibitors of bacterial peptidoglycan synthesis. Chemically modified into imipenem (N-formimidoyl thienamycin), now one of the most clinically important antibiotics, thienamycin is encoded by a thienamycin gene cluster composed of 22 genes (thnA to thnV) from S. cattleya NRRL 8057 genome. Interestingly, the role of all thn-genes has been experimentally demonstrated in the thienamycin biosynthesis, except thnS, despite its annotation as putative β-lactamase. Here, we expressed thnS gene and investigated its activities against various substrates. Our analyses revealed that ThnS belonged to the superfamily of metallo-β-lactamase fold proteins. Compared to known β-lactamases such as OXA-48 and NDM-1, ThnS exhibited a lower affinity and less efficiency toward penicillin G and cefotaxime, while imipenem is more actively hydrolysed. Moreover, like most MBL fold enzymes, additional enzymatic activities of ThnS were detected such as hydrolysis of ascorbic acid, single strand DNA, and ribosomal RNA. ThnS appears as a MBL enzyme with multiple activities including a specialised β-lactamase activity toward imipenem. Thus, like toxin/antitoxin systems, the role of thnS gene within the thienamycin gene cluster appears as an antidote against the produced thienamycin.

Proteins with a metallo-beta-lactamase (MBL) fold have been largely studied in bacteria in the framework of resistance to beta-lactams, but their spectrum of activities is broader. We show here that the giant Tupanvirus also encodes a MBL fold-protein that has orthologs in other giant viruses, a deep phylogenetic root and is clustered with tRNases. This protein is significantly associated with translation components in giant viruses. After expression in Escherichia coli, it was found to hydrolyse nitrocefin, a beta-lactam, and penicillin G. This was inhibited by sulbactam, a beta-lactamase inhibitor. In addition, the tupanvirus MBL fold-protein was not active on single- or double-stranded DNA, but degraded RNAs from bacteria and Acanthamoeba castellanii, the tupanvirus amoebal host. This activity was not neutralized by sulbactam. Overall, our results still broaden the host range of MBL fold-proteins, showing dual beta-lactamase/nuclease activities in giant viruses.

Enzymatic proteins with a metallo-beta-lactamase (MBL) fold have been essentially studied in bacteria for their activity on beta-lactam antibiotics. However, the MBL fold is ancient and highly conserved, and these proteins are capable of cleaving a broad range of substrates. It has recently been shown that MBLs are present in a wide array of cellular organisms, including eukaryotes and archaea. We show here that Tupanvirus deep ocean, a giant virus, also encodes a protein with a MBL fold. Phylogeny showed its clustering with transfer ribonucleases (RNases) and the presence of orthologs in other giant viruses, mainly those harboring the largest sets of translation components. In addition, it suggests an ancient origin for these genes and a transfer between giant viruses and Acanthamoeba spp., a host of many giant viruses. Biologically, after its expression in Escherichia coli, the tupanvirus protein was found to hydrolyse nitrocefin, a chromogenic beta-lactam. We also observed an hydrolysis of penicillin G (10 μg/mL) and detected the metabolite of penicillin G hydrolysis, benzylpenilloic acid. This was inhibited by sulbactam, a beta-lactamase inhibitor. In addition, we tested the degradation of single-stranded DNA, double-stranded DNA, and RNAs, and observed a strong activity on RNAs from seven bacteria with G+C varying from 42% to 67%, and from Acanthamoeba castellanii, the tupanvirus host. This was not inhibited by sulbactam or ceftriaxone. RNase activity was estimated to be 0.45±0.15 mU/mg using a fluorescence-based assay. Our results still broaden the range of hosts of MBL fold proteins and demonstrate that such protein can have dual beta-lactamase/nuclease activities. We suggest that they should be annotated according to this finding to avoid further confusion.

The Acanthamoeba polyphaga mimivirus is the first giant virus ever described, with a 1.2-Mb genome which encodes 979 proteins, including central components of the translation apparatus. One of these proteins, R458, was predicted to initiate translation, although its specific role remains unknown. We silenced the R458 gene using small interfering RNA (siRNA) and compared levels of viral fitness and protein expression in silenced versus wild-type mimivirus. Silencing decreased the growth rate, but viral particle production at the end of the viral cycle was unaffected. A comparative proteomic approach using two-dimensional difference-in-gel electrophoresis (2D-DIGE) revealed deregulation of the expression of 32 proteins in silenced mimivirus, which were defined as up- or downregulated. Besides revealing proteins with unknown functions, silencing R458 also revealed deregulation in proteins associated with viral particle structures, transcriptional machinery, oxidative pathways, modification of proteins/lipids, and DNA topology/repair. Most of these proteins belong to genes transcribed at the end of the viral cycle. Overall, our data suggest that the R458 protein regulates the expression of mimivirus proteins and, thus, that mimivirus translational proteins may not be strictly redundant in relation to those from the amoeba host. As is the case for eukaryotic initiation factor 4a (eIF4a), the R458 protein is the prototypical member of the ATP-dependent DEAD box RNA helicase mechanism. We suggest that the R458 protein is required to unwind the secondary structures at the 5′ ends of mRNAs and to bind the mRNA to the ribosome, making it possible to scan for the start codon. These data are the first experimental evidence of mimivirus translation-related genes, predicted to initiate protein biosynthesis. IMPORTANCE The presence in the genome of a mimivirus of genes coding for many translational processes, with the exception of ribosome constituents, has been the subject of debate since its discovery in 2003. In this work, we focused on the R458 mimivirus gene, predicted to initiate protein biosynthesis. After silencing was performed, we observed that it has no major effect on mimivirus multiplication but that it affects protein expression and fitness. This suggests that it is effectively used by mimivirus during its developmental cycle. Until large-scale genetic manipulation of giant viruses becomes possible, the silencing strategy used here on mimivirus translation-related factors will open the way to understanding the functions of these translational genes.

BACKGROUND: Necrotizing enterocolitis (NEC) is the most common and serious gastrointestinal disorder among preterm neonates. We aimed to assess a specific gut microbiota profile associated with NEC. METHODS: Stool samples and clinical data were collected from 4 geographically independent neonatal intensive care units, over a 48-month period. Thirty stool samples from preterm neonates with NEC (n = 15) and controls (n = 15) were analyzed by 16S ribosomal RNA pyrosequencing and culture-based methods. The results led us to develop a specific quantitative polymerase chain reaction (qPCR) assay for Clostridium butyricum, and we tested stool samples from preterm neonates with NEC (n = 93) and controls (n = 270). We sequenced the whole genome of 16 C. butyricum strains, analyzed their phylogenetic relatedness, tested their culture supernatants for cytotoxic activity, and searched for secreted toxins. RESULTS: Clostridium butyricum was specifically associated with NEC using molecular and culture-based methods (15/15 vs 2/15; P < .0001) or qPCR (odds ratio, 45.4 [95% confidence interval, 26.2-78.6]; P < .0001). Culture supernatants of C. butyricum strains from preterm neonates with NEC (n = 14) exhibited significant cytotoxic activity (P = .008), and we identified in all a homologue of the β-hemolysin toxin gene shared by Brachyspira hyodysenteriae, the etiologic agent of swine dysentery. The corresponding protein was secreted by a NEC-associated C. butyricum strain. CONCLUSIONS: NEC was associated with C. butyricum strains and dysbiosis with an oxidized, acid, and poorly diversified gut microbiota. Our findings highlight the plausible toxigenic mechanism involved in the pathogenesis of NEC.

International audience; Background: Literature reports on ophthalmological manifestations related to tularemia, a zoonose caused by the bacterium Francisella tularensis, largely refer to Parinaud's oculoglandular syndrome, which consists of the association of conjunctivitis with preauricular lymphadenitis. In this paper, we report a case of intraocular inflammation during tularemia infection. Case presentation: A 52-year-old Caucasian man was diagnosed with unilateral uveitis. The uveitis was posterior, with a 2+ vitritis and a large yellowish lesion involving the macula with an overlying sub-retinal detachment, extending inferiorly, and subretinal hemorrhages. Fluorescein angiography showed a late hyperfluorescence with focal vascular leakage. Ultrasound biomicroscopy confirmed the presence of a 3.8 mm parietal granuloma with a few calcifications in the left eye. While extensive work-up eliminated any other infectious and non-infectious etiology, tularemia was diagnosed by advanced serology consisting of two-dimensional Western-immunoblotting. The patient, a hunter, recalled having killed rabbits in the days before the symptoms appeared. Uveitis was rapidly controlled following treatment with doxycycline, yet three years after initiation of the treatment, the patient still complained of loss of vision in the left eye with a central scotoma. Conclusions: Posterior uveitis may be an infrequent manifestation of tularemia infection, and therefore this infection should be considered in the differential diagnosis of intraocular inflammation in areas where F. tularensis is endemic.

Bartonella henselae is an emerging gram-negative facultative intracellular pathogen transmitted via Ctenocephalides felis (cat fleas) or cat scratches. Bartonellosis is present mainly in the form of cat scratch disease (CSD), bacillary angiomatosis and infective endocarditis (IE). The methods used to diagnose B. henselae rely on culturing, immunofluorescent assays and molecular techniques. The objective of the present study was to identify candidate proteins for the serodiagnosis of bartonellosis with the differential discrimination of both clinical scenarios: CSD and IE. For this, an immunoproteomic approach combined with 2-DE, immunoblotting and matrix-assisted laser desorption/ionization time-of-flight MS has been developed. Immunoproteomic profiles of sera collected from patients with CSD and IE were compared with those of blood donors. We identified several candidate proteins as phage-encoding Pap31 protein and an outer membrane protein of BH11510 that, in our view, might be useful for the serodiagnosis of bartonellosis.

Whipple's disease (WD) is a chronic multisystemic infection, caused by the bacterium Tropheryma whipplei. The main clinical presentations are classic WD (CWD) with histologic lesions in the gastrointestinal tract, endocarditis, and isolated neurologic infection. The current strategy for diagnosis remains invasive.The present study aimed to select the protein candidates for serological diagnosis of WD. The first step was to identify candidate proteins by an immunoproteomic approach combining 2-DE using a total extract of a T. whipplei, immunoblotting, and MS. The second step was to validate the discovered biomarkers using a recombinant protein-based ELISA. Serum samples from 18 patients with WD and from 54 control individuals were tested. A sugar ABC transporter, TWT328 (sensitivity (Se) 61%, specificity (Sp) 87%, positive predictive value (PPV) 61%, negative predictive value (NPV) 87%, and positive likelihood ratio (PLR) 4.69) was the best marker for development of serodiagnosis for CWD. We also obtained a reproducible immunoreactive protein pattern for patients with isolated neurological infection due to T. whipplei (Se 100%, Sp 93%, PPV 55.5%, NPV 100%, and PLR 13.51) as an encouraging step towards noninvasive diagnosis of this particular manifestation. Nine recombinant candidates have been successfully screened with serum samples. Results from these ELISA assays skewed with those obtained with immunoblots.

International audience; Relapsing fever borreliae, produced by ectoparasite-borne Borrelia species, cause mild to deadly bacteremia and miscarriage. In the perspective of developing inexpensive assays for the rapid detection of relapsing fever borreliae, we produced 12 monoclonal antibodies (MAbs) against Borrelia crocidurae and characterized the two exhibiting the highest titers. P3A10 MAb reacts with the 35.6-kDa flagellin B (flaB) of B. crocidurae while P6D9 MAb recognizes a 35.1-kDa variable-like protein (Vlp) in B. crocidurae and a 35.2-kDa Vlp in Borrelia duttonii. Indirect immunofluorescence assay incorporating relapsing fever and Lyme group borreliae and 11 blood-borne organisms responsible for fever in West Africa confirmed the reactivity of these two MAbs. Combining these two MAbs in indirect immunofluorescence assays detected relapsing fever borreliae including B. crocidurae in ticks and the blood of febrile Senegalese patients. Both antibodies could be incorporated into inexpensive and stable formats suited for the rapid point-of-care diagnosis of relapsing fever. These first-ever MAbs directed against African relapsing fever borreliae are available for the scientific community to promote research in this neglected field.

Background. Bacterial endocarditis is a serious disease. Surface-enhanced laser desorption/ionization time-of-flight (SELDI-TOF) mass spectrometry (MS) based on serum protein profiling is a powerful approach that can generate biomarkers with diagnostic value. Methods. To identify a protein signature associated with bacterial endocarditis, we retrospectively performed SELDI-TOF MS profiling of serum samples from 88 patients hospitalized because of clinical suspicion of endocarditis. The diagnosis was confirmed by conventional criteria for 34 patients (endocarditis positive) and was excluded for 54 patients (endocarditis negative). Serum samples were incubated with cation-exchange ProteinChip arrays. The protein profiles generated were subjected to biostatistical processing. Results. Fifty-nine samples (23 endocarditis positive and 36 endocarditis negative) were randomly selected for a learning set, with the 29 remaining samples (11 endocarditis positive and 18 endocarditis negative) serving as an independent testing (validation) set. Sixty-six protein peaks were differentially expressed between the endocarditis-positive and the endocarditis-negative patients. By combining partial least squares and logistic regression methods, we built a serum protein model that perfectly discriminated between endocarditis-positive and endocarditis-negative patients. Importantly, when this model was tested on the independent testing set, a correct prediction rate of nearly 90% was demonstrated. Overall, sensitivity, specificity, positive predictive value, and negative predictive value were 94%, 98%, 96%, and 96%, respectively. Conclusions. SELDI-TOF MS profiling revealed a serum signature with high diagnostic potential for endocarditis.

International audience; The study of the human virome is still in its infancy, especially with regard to the viral content of the blood of people who are apparently disease free. In this study, the genome of a new giant virus that is related to the amoeba-infecting pathogen Marseillevirus was recovered from donated blood, using high-throughput sequencing. Viral antigens were identified by an immunoconversion assay. The virus was visualized with transmission electron microscopy and fluorescence in situ hybridization and was grown in human T lymphocytes. Specific antibody reactions were used to identify viral proteins in blood specimens from polymerase chain reactive-positive donors. Finally, we tested 20 blood specimens from additional donors. Three had antibodies directed against this virus, and 2 had circulating viral DNA. This study shows that giant viruses, which are missed by the use of ultrafilters, are part of the human blood virome. The putative pathogenic role of giant viruses in humans remains undefined.

Peptide methionine sulfoxide reductase mediates the reduction of protein sulfoxide methionyl residues back to methionines and could thus be implicated in the antioxidant defence of organisms. Hexagonal crystals of the Escherichia coli enzyme (MsrA) were obtained by the hanging-drop vapour-diffusion technique. They belong to space group P6(5)22, with unit-cell parameters a = b = 102.5, c = 292.3 A, gamma = 120 degrees. A native data set was collected at 1.9 A resolution. Crystals of selenomethionine-substituted MsrA were also grown under the same crystallization conditions. A three-wavelength MAD experiment has led to the elucidation of the positions of the Se atoms and should result in a full structure determination.

International audience