Anne-Béatrice Blanc-Potard

People living with HIV (PLWH) have a higher risk of developing other diseases, such as bacterial pneumonia. Pseudomonas aeruginosa is a common cause of pneumonia in humans. The most virulent strains produce ExoU, which is a key effector of P. aeruginosa cytotoxicity. Upon injection into the target cell cytosol by the type III secretion system (T3SS), ExoU binds to PI(4,5)P ₂ on the inner leaflet of the plasma membrane. ExoU phospholipase activity then induces a loss of plasma membrane integrity, rapidly leading to cell death. HIV-1-Tat is secreted by HIV-infected cells, leading to nanomolar concentrations of Tat in the sera of PLWH, even under antiretroviral therapy. Circulating Tat is endocytosed by uninfected cells, translocates to the cytosol, and binds to PI(4,5)P ₂ at the plasma membrane. In uninfected cells only, Tat is palmitoylated, enabling Tat to become resident on PI(4,5)P ₂ . Here, we found that Tat interferes with the recruitment of ExoU by PI(4,5)P ₂ , thereby protecting primary human macrophages from the toxicity of exoU -positive P. aeruginosa strains. Tat nevertheless enhances the toxicity of exoU -deficient strains of P. aeruginosa toward macrophages. HIV Tat might, therefore, modulate the sensitivity of PLWH toward P. aeruginosa strains depending on whether they own the key type III effector exoU . IMPORTANCE: Pseudomonas aeruginosa is a common cause of pneumonia in humans and a major nosocomial pathogen. People living with HIV (PLWH), even under antiretroviral therapy, are at risk of developing infectious diseases, such as bacterial pneumonia. The T3SS plays an important role in P. aeruginosa virulence. The T3SS can inject four effectors into the target cell cytosol, including the key effector ExoU that binds to PI(4,5)P2 on the inner leaflet of the plasma membrane. The potent phospholipase activity of ExoU rapidly induces cell death. HIV-Tat is present in the serum of PLWH. Circulating Tat enters cells and binds PI(4,5)P ₂ with a very high affinity. Our results indicate that HIV-Tat interferes with the recruitment of ExoU by PI(4,5)P ₂ , thereby protecting macrophages from strains of P. aeruginosa that express ExoU. Tat, nevertheless, increased macrophage sensitivity to exoU-negative strains. HIV Tat could, therefore, modulate PLWH susceptibility to P. aeruginosa .

The MgtC virulence factor is important during the intramacrophage stage in both classical intracellular pathogens, such as Salmonella Typhimurium, and in extracellular bacteria that transiently encounter intracellular environments during infection, such as Pseudomonas aeruginosa . In these different pathogens, mgtC expression is induced in vitro by magnesium ion depletion, a condition reported to mimic the macrophage environment. Here, we developed an unstable GFP reporter system to monitor in real time the transcriptional activation of the P. aeruginosa mgtC promoter. After in vitro validation in magnesium-defined media, this reporter system allowed visualization of the mgtC promoter induction in a subset of bacteria when P. aeruginosa localized inside cultured macrophages. In addition, although rare under our experimental conditions, in vivo activation of the mgtC promoter was observed for the first time within macrophages of live, infected zebrafish larvae, a cutting-edge vertebrate model for real-time imaging. While MgtC regulation in Salmonella is mediated by the magnesium-responsive PhoPQ two-component system, its regulation in P. aeruginosa remained unknown. The use of mutant strains for two-component regulatory systems revealed that the PhoP regulator, but not by its cognate sensor PhoQ, was required to activate P. aeruginosa MgtC expression in vitro . Unexpectedly, CbrAB, a two-component system specific to Pseudomonas species, was also involved in P. aeruginosa MgtC regulation. Both PhoP and CbrB regulatory proteins were found to directly bind the mgtC promoter, supporting a dual transcriptional control. These findings reveal substantial differences in mgtC gene regulation in different bacterial pathogens, reflecting distinct strategies to drive appropriate expression of a shared virulence factor involved in macrophage adaptation. IMPORTANCE: The adaptation of bacterial pathogens to the host intracellular microenvironment requires tight and rapid regulation of specific genes, and investigating the in vivo transcriptional dynamics of such genes is a major challenge. Here, we focused on the expression of mgtC , a gene important for adaptation to the intramacrophage environment in classical intracellular pathogens, such as Salmonella Typhimurium, and bacteria with a transient intracellular lifestyle, such as Pseudomonas aeruginosa . An unstable GFP reporter system was designed to monitor the transcriptional dynamics of P. aeruginosa mgtC . The use of this reporter system in a state-of-the-art vertebrate model for live imaging, the zebrafish embryo, allowed in vivo tracking of P. aeruginosa mgtC promoter activation inside macrophages in a living host. Furthermore, the expression of P. aeruginosa mgtC was found to be regulated through a mechanism distinct from that of Salmonella MgtC, since it involves the PhoP regulatory protein, but not the PhoQ sensor, and the Pseudomonas -specific CbrAB two-component system, reflecting diverse, finely tuned strategies to control a virulence factor shared by several major human pathogens.

People living with HIV (PLWH) have a higher risk of developing other diseases, such as bacterial pneumonia. While Pseudomonas aeruginosa (PAE) is a common cause of pneumonia in humans, PLWHs are infrequently infected by PAE. The reasons for this relative resistance of PLWH to PAE are not known. The most virulent PAE strains produce ExoU which is a key effector of PAE cytotoxicity. Upon injection into the target cell cytosol by the type III secretion system, ExoU binds to PI(4,5)P ₂ on the inner leaflet of the plasma membrane. Its phospholipase activity then induces a loss of plasma membrane integrity, rapidly leading to cell death. HIV-Tat is secreted by HIV-infected cells, leading to nanomolar concentrations of Tat in the sera of PLWH, even under antiretroviral therapy. Circulating Tat can be endocytosed by uninfected cells, translocate to the cytosol and bind to PI(4,5)P ₂ at the plasma membrane. In uninfected cells only, Tat is palmitoylated, enabling Tat to become resident on PI(4,5)P ₂ . We found that Tat can interfere with the recruitment of ExoU by PI(4,5)P ₂ , thereby protecting macrophages from PAE toxicity. HIV Tat could therefore be involved in the relative protection of PLWH against the most aggressive PAE isolates and their ExoU type III effector. Tat nevertheless enhances the toxicity of ExoU-deficient PAE strains toward macrophages.

Investigating the interplay between polymicrobial colonization and host response in the context of chronic infections is a complex issue. The interaction between Staphylococcus aureus and Pseudomonas aeruginosa, which frequently co-infect patients with chronic diseases like cystic fibrosis (CF), can be either competitive or coexistent. How these interaction states can influence the host response remains an open question for which relevant infection models are needed. In this study, we investigated co-infections by P. aeruginosa and S. aureus in zebrafish (Danio rerio), a non-mammalian model well established for the study of the host innate immune response. Using a wound infection method and a trio of strains co-isolated from a CF patient, we provide an in vivo co-infection model that recapitulates the competitive and coexistent bacterial interactions observed in vitro. The inflammatory response, monitored through the expression of specific cytokines at the infection site or in the whole larvae, was stronger with P. aeruginosa than S. aureus in the context of mono-infection. Upon co-infection, a competitive or coexistent interaction between P. aeruginosa and S. aureus appeared to slightly modulate this response. Moreover, the RT-qPCR profile of cytokine response observed in zebrafish larvae was similar to the one observed after infection of human lung epithelial cells. Thus, the zebrafish embryo appears as a relevant model to study persistent co-infection with P. aeruginosa and S. aureus, offering unique opportunities to address the host response in this polymicrobial context.

Pseudomonas aeruginosa is a major human pathogen, particularly in chronic wound infections and chronic pulmonary infections (especially in patients with cystic fibrosis (CF)). Chronic bacterial infections are refractory to antibiotic treatments and there is an urgent need for in vivo chronic infection models amenable to drug screening to develop efficient therapies. Here, we describe a protocol of infection by a P. aeruginosa clinical isolate from a CF patient, expressing constitutively the Green Fluorescent Protein (GFP), for generating a persistent wound infection in zebrafish larvae. Tail fin-injured embryos are immersed in a bacterial suspension for 1.5 h, washed, and monitored for bacterial load for 3 days. The bacterial burden was quantified daily by counting fluorescent colony-forming units (CFU) from lysed infected larvae, which allowed a persistent infection. Moreover, persistent P. aeruginosa bacteria were refractory to antibiotic treatment. This novel in vivo model of persistent P. aeruginosa infection offers opportunities to evaluate the efficacy of innovative treatments against chronic infections.

Pseudomonas aeruginosa is a major human pathogen, particularly in chronic wound infections and chronic pulmonary infections (especially in patients with cystic fibrosis (CF)). Chronic bacterial infections are refractory to antibiotic treatments and there is an urgent need for in vivo chronic infection models amenable to drug screening to develop efficient therapies. Here, we describe a protocol of infection by a P. aeruginosa clinical isolate from a CF patient, expressing constitutively the Green Fluorescent Protein (GFP), for generating a persistent wound infection in zebrafish larvae. Tail fin-injured embryos are immersed in a bacterial suspension for 1.5 h, washed, and monitored for bacterial load for 3 days. The bacterial burden was quantified daily by counting fluorescent colony-forming units (CFU) from lysed infected larvae, which allowed a persistent infection. Moreover, persistent P. aeruginosa bacteria were refractory to antibiotic treatment. This novel in vivo model of persistent P. aeruginosa infection offers opportunities to evaluate the efficacy of innovative treatments against chronic infections.

Persistent bacterial infections evade host immunity and resist antibiotic treatments through various mechanisms that are difficult to evaluate in a living host. Pseudomonas aeruginosa is a main cause of chronic infections in patients with cystic fibrosis (CF) and wounds. Here, by immersing wounded zebrafish embryos in a suspension of P. aeruginosa isolates from CF patients, we established a model of persistent infection that mimics a murine chronic skin infection model. Live and electron microscopy revealed persisting aggregated P. aeruginosa inside zebrafish cells, including macrophages, at unprecedented resolution. Persistent P. aeruginosa exhibited adaptive resistance to several antibiotics, host cell permeable drugs being the most efficient. Moreover, persistent bacteria could be partly re-sensitized to antibiotics upon addition of anti-biofilm molecules that dispersed the bacterial aggregates in vivo. Collectively, this study demonstrates that an intracellular location protects persistent P. aeruginosa in vivo in wounded zebrafish embryos from host innate immunity and antibiotics, and provides new insights into efficient treatments against chronic infections.

Persistent bacterial infections evade host immunity and resist antibiotic treatments through various mechanisms that are difficult to evaluate in a living host. Pseudomonas aeruginosa is a main cause of chronic infections in patients with cystic fibrosis (CF) and wounds. Here, by immersing wounded zebrafish embryos in a suspension of P. aeruginosa isolates from CF patients, we established a model of persistent infection that mimics a murine chronic skin infection model. Live and electron microscopy revealed persisting aggregated P. aeruginosa inside zebrafish cells, including macrophages, at unprecedented resolution. Persistent P. aeruginosa exhibited adaptive resistance to several antibiotics, host cell permeable drugs being the most efficient. Moreover, persistent bacteria could be partly re-sensitized to antibiotics upon addition of anti-biofilm molecules that dispersed the bacterial aggregates in vivo. Collectively, this study demonstrates that an intracellular location protects P. aeruginosa in vivo from host innate immunity and antibiotics, and provides new insights into efficient treatments against chronic infections.

Pseudomonas aeruginosa often colonizes immunocompromised patients, causing acute and chronic infections. This bacterium can reside transiently inside cultured macrophages, but the contribution of the intramacrophic stage during infection remains unclear. MgtC and OprF have been identified as important bacterial factors when P. aeruginosa resides inside cultured macrophages. In this study, we showed that P. aeruginosa mgtC and oprF mutants, particular the latter one, had attenuated virulence in both mouse and zebrafish animal models of acute infection. To further investigate P. aeruginosa pathogenesis in zebrafish at a stage different from acute infection, we monitored bacterial load and visualized fluorescent bacteria in live larvae up to 4 days after infection. Whereas the attenuated phenotype of the oprF mutant was associated with a rapid elimination of bacteria, the mgtC mutant was able to persist at low level, a feature also observed with the wild-type strain in surviving larvae. Interestingly, these persistent bacteria can be visualized in macrophages of zebrafish. In a short-time infection model using a macrophage cell line, electron microscopy revealed that internalized P. aeruginosa wild-type bacteria were either released after macrophage lysis or remained intracellularly, where they were localized in vacuoles or in the cytoplasm. The mgtC mutant could also be detected inside macrophages, but without causing cell damage, whereas the oprF mutant was almost completely eliminated after phagocytosis, or localized in phagolysosomes. Taken together, our results show that the main role of OprF for intramacrophage survival impacts both acute and persistent infection by this bacterium. On the other hand, MgtC plays a clear role in acute infection but is not essential for bacterial persistence, in relation with the finding that the mgtC mutant is not completely eliminated by macrophages.

Escherichia coli isolated from meat of different animal species may harbour antimicrobial resistance genes and may thus be a threat to human health. The objectives of this study were to define antimicrobial resistance genes in E. coli isolates from pork, beef, chicken- and turkey meat and analyse whether their resistance genotypes associated with phylogenetic groups or meat species. A total number of 313 E. coli samples were isolated using standard cultural techniques. In 98% of resistant isolates, a dedicated resistance gene could be identified by PCR. Resistance genes detected were tet(A) and tet(B) for tetracycline resistance, strA and aadA1 for streptomycin resistance, sulI and sulII for resistance against sulphonamides, dfr and aphA for kanamycin resistance and bla_TEM for ampicillin resistance. One stx1 harbouring E. coli isolated from pork harboured the tet(A) gene and belonged to phylogenetic group B2, whilst another stx1 positive isolate from beef was multi-resistant and tested positive for bla_TEM,aphA, strA–B, sulII, and tet(A) and belonged to phylogenetic group A. In conclusion, the distribution of resistance elements was almost identical and statistically indifferent in isolates of different meat species. Phylogenetic groups did not associate with the distribution of resistance genes and a rather low number of diverse resistance genes were detected. Most E. coli populations with different resistance genes against one drug often revealed statistically significant different MIC values.