The DEAD-box RNA helicase CshA is required for fatty acid homeostasis inStaphylococcus aureus

Staphylococcus aureusis an opportunistic pathogen that can grow in a wide array of conditions: on abiotic surfaces, on the skin, in the nose, in planktonic or biofilm forms and can cause many type of infections. Consequently,S.aureusmust be able to adapt rapidly to these changing growth conditions, an ability largely driven at the posttranscriptional level. RNA helicases of the DEAD-box family play an important part in this process. In particular, CshA, which is part of the degradosome, is required for the rapid turnover of certain mRNAs and its deletion results in cold-sensitivity. To understand the molecular basis of this phenotype, we conducted a large genetic screen isolating 82 independent suppressors of cold growth. Full genome sequencing revealed the fatty acid synthesis pathway affected in many suppressor strains. Consistent with that result, sublethal doses of triclosan, a FASII inhibitor, can partially restore growth of acshAmutant in the cold. Overexpression of the genes involved in branched-chain fatty acid synthesis was also able to suppress the cold-sensitivity. Using gas chromatography analysis of fatty acids, we observed an imbalance of straight and branched-chain fatty acids in thecshAmutant, compared to the wild-type. This imbalance is compensated in the suppressor strains. Thus, we reveal for the first time that the cold sensitive growth phenotype of a DEAD-box mutant can be explained, at least partially, by an improper membrane composition. The defect correlates with an accumulation of the pyruvate dehydrogenase complex mRNA, which is inefficiently degraded in absence of CshA. We propose that the resulting accumulation of acetyl-CoA fuels straight-chained fatty acid production at the expense of the branched ones. Strikingly, addition of acetate into the medium mimics thecshAdeletion phenotype, resulting in cold sensitivity suppressed by the mutations found in our genetic screen or by sublethal doses of triclosan. Author summary DEAD-box RNA helicases are highly conserved proteins found in all domains of life. By acting on RNA secondary structures they determine the fate of RNA from transcription to degradation. Bacterial DEAD-box RNA helicases are not essential under laboratory conditions but are required for fitness and under stress conditions. Whereas many DEAD-box protein mutants display a cold sensitive phenotype, the underlying mechanisms have been studied only in few cases and found to be associated with ribosome biogenesis. We aimed here to elucidate the cold sensitivity of acshAmutant in the Gram-positive opportunist pathogenStaphylococcus aureus. Our study revealed for the first time that part of the cold sensitivity is related to the inability of the bacterium to adapt the cytoplasmic membrane to lower temperatures. We propose that straight-chain fatty acid synthesis, reduced to sustain growth at lower temperature, is maintained due to inefficient turn-over of the pyruvate dehydrogenase mRNA, leading to elevated acetyl-CoA levels. This study allowed us to unravel at least in part the cold sensitive phenotype and to show that the pyruvate dehydrogenase activity plays an important function in the regulation of fatty acid composition of the membrane, a process that remains poorly understood in Gram-positive bacteria.

Role and regulation of the melanization reaction in Drosophila immune response

Olivier Binggeli

In arthropods, the melanization reaction consists in the rapid synthesis of melanin at the site of infection and injury. A key enzyme in the melanization process is phenoloxidase (PO), which catalyzes the oxidation of phenols to quinones that subsequently polymerize into melanin. PO is synthesized under an inactive form called pro-PO (PPO) that is activated upon cleavage by a terminal protease of a serine protease cascade. The main objective of my PhD thesis is to understand the role and regulation of the melanization reaction in the Drosophila immune response. The Drosophila genome encodes three PPOs, two of which, PPO1 and PPO2, are produced by crystal cells, a larval-specific type of hemocyte. I generated flies carrying null mutations in PPO1 and PPO2 as well as PPO1, PPO2 double mutant flies. My results show that both PPO1 and PPO2 contribute to the PO activity in the hemolymph induced by septic injury. While PPO1 is involved in the early delivery of PO activity, PPO2 is stored in crystalline form in the crystal cells and can be deployed at a later phase of the immune response. My genetic analysis also reveals an important role of PPO1 and PPO2 in the resistance of flies to infection with Gram-positive bacteria and fungi. This study definitively establishes the importance of melanization in insect host defense. Serine proteases regulate many immune reactions, including blood clotting, the complement system in mammals, and melanization and Toll pathway activation in insects. A second objective of my thesis is to identify new serine proteases or serine protease inhibitors (serpins) participating in the Drosophila immune response. I performed an RNAi screen to identify genes encoding serine protease or serpins whose inactivation caused an immune phenotype. In parallel, I identified or generated mutations affecting some serine proteases and serpins and described their immune phenotype. This led to the identification of SPH9, a gene strongly induced by microbial infection, which encodes a serine protease homologue that negatively regulates the melanization reaction. Together, my results provide new insights on the melanization reaction in Drosophila.

EPFL2013

Mechanistic aspects of DNA uptake in naturally competent Vibrio cholerae

Patrick Martin Seitz

The physiological state of competence for natural transformation allows bacteria to take up free DNA from the environment and recombine it into their genome. As one of three modes of horizontal gene transfer, natural transformation has hence contributed substantially to the enormous diversity and plasticity of bacterial genomes. With its potential to promote the spread of antibiotic resistances and virulence factors of a variety of pathogens, natural transformation also has an important impact on human health. Even though conserved components required to undergo natural transformation have been identified in many bacterial species, our knowledge on the underlying mechanism of DNA transport remains very limited. To better understand this aspect, we established a first model of DNA uptake in the naturally competent human pathogen Vibrio cholerae. We showed that at least twenty proteins are required for efficient uptake, protection and recombination of transforming DNA in a non-species-specific manner. 14 of these proteins are needed to produce a type IV pilus, which we were able to visualize for the first time. We showed that this competence pilus is not restricted to cell poles, as has been shown for other competent bacterial species (e.g. Bacillus subtilis). Furthermore, we compared the position of this pilus to the localization of other competence proteins and determined their role in the assembly of a surface exposed pilus fiber. While this type IV pilus is important for efficient DNA uptake, we also observed rare transformation events in its absence. In contrast, three pilus-unrelated proteins that are likely involved in the transport process were absolutely required for transformation. Two of them were implicated in DNA transport across the inner membrane. Deletion of the corresponding genes led to a strong accumulation of transforming DNA within the periplasm. However, one of them, ComEA, was essential for DNA uptake across the outer membrane, independently of competence pili. We further characterized the role of this protein by showing that it is localized to the periplasm of competent V. cholerae cells and that it binds to DNA in vivo. Based on our results, we proposed that ComEA directly contributes to DNA transport. Although we showed that DNA uptake across the outer membrane and translocation of DNA into the cytoplasm are functionally uncoupled, our results suggest that the two events coincide spatially. In summary, these findings allow us to present a first mechanistic model of the DNA uptake process in V. cholerae. Many aspects of this model likely apply to other naturally competent bacteria, and therefore represent an important contribution to the general understanding of this basic biological process.

EPFL2014

Characterization of the electron-accepting part of the organohalide respiration chain of Dehalobacter and Desulfitobacterium

Lorenzo Cimmino

Organohalide respiration (OHR) is a bacterial anaerobic process that use halogenated compounds, e.g. tetrachloroethene (PCE), as terminal electron acceptors. D. restrictus strain PER-K23, an obligate OHR bacterium (OHRB), and D. hafniense strain TCE1, a bacterium with a versatile metabolism, harbour the pceABCT gene cluster, which represents our model system for the study of PCE respiration. To date, the function of PceA, the key enzyme in the process, and PceT, the molecular chaperone for PceA maturation, are well defined. However, the role of PceB and PceC are still not elucidated and the biochemistry of OHR electron transfer is still relatively elusive. Based on the genetic composition of the pce gene cluster, the hypothesis that PceB and PceC may play a role in electron transfer in the metabolism of organohalide respiration is tempting but the question remains largely unanswered. In the present Ph.D. thesis, a multilevel study aiming at characterizing the electron-accepting part of the respiratory chain of OHRB will be presented. The investigation was assessed at molecular level by the deciphering of the stoichiometry of pceABCT individual gene products, and at physiological and biochemical levels, where the characterization of the membrane PceA-containing protein complex of both obligate and facultative OHRB was the main focus. The stoichiometry analysis at RNA level revealed that the pce gene cluster is an operon with, however, a level of transcription that differs for individual genes, an observation that could be explained by post-transcriptional events. At proteomic level, an apparent 2:1 stoichiometry of PceA and PceB was obtained in the membrane fraction, while low abundance of PceC in comparison to the other two proteins was observed. In the soluble fraction, a 1:1 stoichiometry of PceA and PceT was identified. Furthermore, a combination of Clear-Native PAGE gel and a in-gel PceA enzymatic activity assay were applied to identify the RDH complex from the membrane of D. restrictus and D. hafniense strain TCE1. The results revealed an active RDH complex in the membrane extract of both organisms with an estimated molecular mass of 180 kDa, while no RDH activity could be detected in the soluble fraction. Furthermore, immunoblotting for PceA on CN-PAGE gel revealed the presence of a second, however inactive, PceA-containing complex with a molecular mass of 670 kDa, which was confirmed by MS analysis to be largely dominated by the molecular chaperone GroEL alongside with PceA and likely representing the GroEL maturation complex. The RDH complex was purified from the membrane extract by chromatography. The obtained fractions were analysed by LC-MS/MS showing unambiguously the presence of PceA and PceB. Furthermore, preliminary cryo-EM analysis led to propose a 3D reconstruction of the RDH complex, revealing the likely presence of a Pce(AB)2 complex. In summary, this study represents a road-map for the identification of RDH complexes from OHRB. The identification of PceA associated with the GroEL maturation complex raised new questions about the biosynthetic pathway of PceA and invites to consider the involvement of GroEL into the maturation of the key enzyme in organohalide respiration. Finally the preliminary results obtained via cryo-EM analysis set the basis for further investigation on the structure of the RDH complex and open new horizons towards the elucidation of the electron transfer chain involved in the reduction of PCE.

EPFL2022