Caractérisation de phages tempérés et évaluation de leurs impacts sur le phénotype bactérien de clostridium difficile

Clostridium difficile est un pathogène entérique qui cause d'importantes infections nosocomiales dont le traitement est parfois problématique. Il n'existe, à l'heure actuelle, que deux antibiotiques approuvés pour traiter les infections à C. difficile et le taux de rechute est assez important. Ce projet a initialement visé à isoler et caractériser des phages virulents contre C. difficile en vue de les utiliser en phagothérapies comme outils thérapeutiques alternatifs. Les eaux usées et les selles de patients infectés par C. difficile ont été utilisées pour isoler et détecter les phages virulents. Or, quatre phages différents (9MMPOI-O4) ont été isolés mais aucun de ces phages ne s'est révélé être virulent. Les quatre phages tempérés ont donc été caractérisés et leur impact a été évalué sur quelques phénotypes bactériens chez C. difficile dont la motilité et la production des toxines A et B. La caractérisation morphologique des phages (pMMPOl-04 a permis de déterminer que ceux-ci appartiennent à la familles des Myoviridae alors que la caractérisation génomique a permis de démontrer que certains de ces phages sont assez différents entre eux mais également par rapport aux autres phages tempérés, isolés et caractérisés dans la littérature. De façon générale, les phages (pMMPOl-04 ne semblent pas s'induire spontanément de manière plus importante mais suggère que la présence de certains antibiotiques pourrait augmenter l'induction de certains de ces phages. L'impact des phages (pMMPOl-04 sur la motilité chez C. difficile n'a pas démontré que ceux-ci avaient un rôle à jouer sur ce phénotype. Par contre, certains des phages (pMMP semblent augmenter ou diminuer la production en toxines A et B. Les résultats de nos travaux indiquent donc que certains des phages caractérisés présentent des différences importantes qui suggèrent une grande diversité parmi les phages tempérés chez C. difficile. De plus, certains des phages cpMMP auraient la capacité de participer aux transferts horizontaux de matériels génétiques et d'affecter la régulation de certains facteurs de virulence chez C. difficile tel que la production en toxines A et B. Évidemment, des travaux supplémentaires seront nécessaires pour confirmer la modification du phénotype de production en toxines par ces phages mais également sur d'autres phénotypes associés aux autres facteurs de virulence de cette bactérie. [Symboles non conformes

Cleavage of a Neuroinvasive Human Respiratory Virus Spike Glycoprotein by Proprotein Convertases Modulates Neurovirulence and Virus Spread within the Central Nervous System.

International audienceHuman coronaviruses (HCoV) are respiratory pathogens that may be associated with the development of neurological diseases, in view of their neuroinvasive and neurotropic properties. The viral spike (S) glycoprotein is a major virulence factor for several coronavirus species, including the OC43 strain of HCoV (HCoV-OC43). In an attempt to study the role of this protein in virus spread within the central nervous system (CNS) and neurovirulence, as well as to identify amino acid residues important for such functions, we compared the sequence of the S gene found in the laboratory reference strain HCoV-OC43 ATCC VR-759 to S sequences of viruses detected in clinical isolates from the human respiratory tract. We identified one predominant mutation at amino acid 758 (from RRSR↓ G758 to RRSR↓R758), which introduces a putative furin-like cleavage (↓) site. Using a molecular cDNA infectious clone to generate a corresponding recombinant virus, we show for the first time that such point mutation in the HCoV-OC43 S glycoprotein creates a functional cleavage site between the S1 and S2 portions of the S protein. While the corresponding recombinant virus retained its neuroinvasive properties, this mutation led to decreased neurovirulence while potentially modifying the mode of virus spread, likely leading to a limited dissemination within the CNS. Taken together, these results are consistent with the adaptation of HCoV-OC43 to the CNS environment, resulting from the selection of quasi-species harboring mutations that lead to amino acid changes in viral genes, like the S gene in HCoV-OC43, which may contribute to a more efficient establishment of a less pathogenic but persistent CNS infection. This adaptative mechanism could potentially be associated with human encephalitis or other neurological degenerative pathologies

Caractérisation de phages tempérés et évaluation de leurs impacts sur le phénotype bactérien de clostridium difficile

Clostridium difficile est un pathogène entérique qui cause d'importantes infections nosocomiales dont le traitement est parfois problématique. Il n'existe, à l'heure actuelle, que deux antibiotiques approuvés pour traiter les infections à C. difficile et le taux de rechute est assez important. Ce projet a initialement visé à isoler et caractériser des phages virulents contre C. difficile en vue de les utiliser en phagothérapies comme outils thérapeutiques alternatifs. Les eaux usées et les selles de patients infectés par C. difficile ont été utilisées pour isoler et détecter les phages virulents. Or, quatre phages différents (9MMPOI-O4) ont été isolés mais aucun de ces phages ne s'est révélé être virulent. Les quatre phages tempérés ont donc été caractérisés et leur impact a été évalué sur quelques phénotypes bactériens chez C. difficile dont la motilité et la production des toxines A et B. La caractérisation morphologique des phages (pMMPOl-04 a permis de déterminer que ceux-ci appartiennent à la familles des Myoviridae alors que la caractérisation génomique a permis de démontrer que certains de ces phages sont assez différents entre eux mais également par rapport aux autres phages tempérés, isolés et caractérisés dans la littérature. De façon générale, les phages (pMMPOl-04 ne semblent pas s'induire spontanément de manière plus importante mais suggère que la présence de certains antibiotiques pourrait augmenter l'induction de certains de ces phages. L'impact des phages (pMMPOl-04 sur la motilité chez C. difficile n'a pas démontré que ceux-ci avaient un rôle à jouer sur ce phénotype. Par contre, certains des phages (pMMP semblent augmenter ou diminuer la production en toxines A et B. Les résultats de nos travaux indiquent donc que certains des phages caractérisés présentent des différences importantes qui suggèrent une grande diversité parmi les phages tempérés chez C. difficile. De plus, certains des phages cpMMP auraient la capacité de participer aux transferts horizontaux de matériels génétiques et d'affecter la régulation de certains facteurs de virulence chez C. difficile tel que la production en toxines A et B. Évidemment, des travaux supplémentaires seront nécessaires pour confirmer la modification du phénotype de production en toxines par ces phages mais également sur d'autres phénotypes associés aux autres facteurs de virulence de cette bactérie. [Symboles non conformes

Neuroinvasive and neurotropic human respiratory coronaviruses: potential neurovirulent agents in humans.

International audienceIn humans, viral infections of the respiratory tract are a leading cause of morbidity and mortality worldwide. Several recognized respiratory viral agents have a neuroinvasive capacity since they can spread from the respiratory tract to the central nervous system (CNS). Once there, infection of CNS cells (neurotropism) could lead to human health problems, such as encephalitis and long-term neurological diseases. Among the various respiratory viruses, coronaviruses are important pathogens of humans and animals. Human Coronaviruses (HCoV) usually infect the upper respiratory tract, where they are mainly associated with common colds. However, in more vulnerable populations, such as newborns, infants, the elderly, and immune-compromised individuals, they can also affect the lower respiratory tract, leading to pneumonia, exacerbations of asthma, respiratory distress syndrome, or even severe acute respiratory syndrome (SARS). The respiratory involvement of HCoV has been clearly established since the 1960s. In addition, for almost three decades now, the scientific literature has also demonstrated that HCoV are neuroinvasive and neurotropic and could induce an overactivation of the immune system, in part by participating in the activation of autoreactive immune cells that could be associated with autoimmunity in susceptible individuals. Furthermore, it was shown that in the murine CNS, neurons are the main target of infection, which causes these essential cells to undergo degeneration and eventually die by some form of programmed cell death after virus infection. Moreover, it appears that the viral surface glycoprotein (S) represents an important factor in the neurodegenerative process. Given all these properties, it has been suggested that these recognized human respiratory pathogens could be associated with the triggering or the exacerbation of neurological diseases for which the etiology remains unknown or poorly understood

Mutation in the spike glycoprotein of mutant virus delays viral spreading compared to the reference strain in mixed primary CNS cultures from BALB/c mice.

<p>Mixed primary cultures from BALB/c mice brain were infected with rOC/ATCC or rOC/S_G758R at MOI 0.03. Viral spread was evaluated at 8, 24 and 48 hpi. Neurons were stained in green with a mAb against microtubule-associated protein 2 (MAP2) antibody and the S viral glycoprotein in red, was detected with a rabbit antiserum. Results are representative of three independent experiments. Magnification 200x.</p

A decreased neurovirulence is observed for rOC/S_G758R variant in 21 day-old BALB/c female mice infected by the intracerebral route.

<p>21 day-old BALB/c mice received 10^2.5 TCID₅₀/10μL of rOC/ATCC or rOC/S_G758R or PBS by the IC route. (A) Survival curves of mice in % during 21 day post-infection. Difference between the two virus variants was significant (* P≤0.05). (B) BALB/c mice were weighed every 2 days during 21 dpi to estimate weight variations, which were expressed in %, compared to day 0 (100%). Differences were significant (*** P≤0.001) when the three conditions (rOC/ATCC, rOC/S_G758R or PBS) were compared between 9 and 11 dpi. Evaluation of the clinical scores (percentage of mice at each level of the scale) of mice infected by rOC/ATCC (C) or rOC/S_G758R (D) based on neurological symptoms described in clinical score scale between level 0 and 3 (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005261#sec011" target="_blank">Materials and Methods</a> section). Production of infectious viral particles was measured in brains (E) and spinal cords (F) every 2 days for 21 dpi. LOD represents the Limit of Detection of infectious viral particles. Results shown are the mean values (with standard deviations) of three independent experiments.</p

A delay in viral spread is observed in brains of rOC/S_G758R -infected mice compared to rOC/ATCC after intracerebral infection in 21 day-old BALB/c female mice.

<p>Histological examination of virus spread within the brain. 21 day-old BALB/c mice received 10^2.5TCID₅₀/10μL of rOC/ATCC or rOC/S_G758R, or PBS by the IC route. Detection of viral antigens in the olfactory bulb (A) or in the hippocampus (B) of infected mice at 5 and 7 dpi at magnitude X40. Black arrows indicate viral particles staining for the S protein.</p

Both variants rOC/ATCC and rOC/S_G758R are neuroinvasive and neurovirulent in 10 day-old BALB/c mice infected by the intranasal route.

<p>10-day old BALB/c mice received 10^3.25 TCID₅₀/10μL of rOC/ATCC or rOC/S_G758R or PBS by the IN route. (A) Survival curves of mice in percentage (%) during 21 days post-infection. Difference between the two variants was significant (** P≤0.01) (B) Surviving BALB/c mice were weighed every 2 days after infection during 21 dpi to estimate weight variations. The weight variation was expressed in %, compared to day 0, which was set at 100%. Production of infectious viral particles was measured in brains (C) and spinal cords (D) every 2 days for 21 dpi. LOD represents the Limit of Detection of infectious viral particles. Results shown are the mean values (with standard deviations) of three independent experiments.</p

The S glycoprotein harboring a predominant point mutation found in clinical isolates was cleaved more efficiently in supernatant of CNS cells.

<p>Mixed primary cultures from BALB/c mice brain were infected with rOC/ATCC or rOC/S_G758R at MOI 0.03. Kinetics of infectious virus production in cell-associated (A) and in cell culture supernatant (free virus) (B) was performed. Release of free virus in the supernatant was significantly higher for rOC/S_G758R compared to rOC/ATCC (* P≤0.05). (C) Western blot analysis of whole cell lysates (C) or cell culture supernatant (D) (10 μg of proteins) revealed the presence of the uncleaved form of the S glycoprotein (180 kDa), and of a cleaved form at around 100 kDa (S1/S2). Results shown are the mean values (with standard deviations) of three independent experiments.</p

A delay in viral spread is observed in brain of rOC/S_G758R -infected mice compared to rOC/ATCC after intranasal inoculation in 10 day-old BALB/c mice.

<p>Histological examination of virus spread within the brain of 10-day old BALB/c mice infected with 10^3.25 TCID₅₀/10μL of rOC/ATCC or rOC/S_G758R, or PBS by the IN route. (A) Detection of viral antigens in the olfactory bulb of infected mice at 5 and 7 dpi. (B) Detection of viral antigens in the hippocampus of infected mice at 7 and 9 dpi. Magnification 40x.</p