Molecular Microbiology最新文献

Trypanosoma cruzi, a flagellated protozoan, is the causative agent of Chagas disease. The parasite has developed various mechanisms to get through its intricate life cycle and adapt to different evolutionary phases. T. cruzi proliferates in the insect vector's digestive tract as an epimastigote form, encountering fluctuating nutrient availability and oxidative stress caused by the digestion of red blood cells from the mammalian host blood meal. To unravel how the parasite's metabolism adapts to these changing conditions, we conducted an analysis of the chemical species present in epimastigote forms. This involved comparing cultured parasites with those subjected to nutritional deficiency or oxidative stress using untargeted metabolomics. We looked at 21 samples: seven biological copies of parasites that were actively growing, seven samples that were put in a medium without nutrients for 3 h, and seven samples that were treated with glucose oxidase for 30 min to make H₂O₂ continuously. Importantly, in all conditions, parasite viability was maintained when the samples were collected. Upon nutrient removal, we observed a substantial decrease in amino acids and carbohydrate metabolites, accompanied by the accumulation of fatty acids and steroids, with the predominance of inositol and sphingolipid metabolism, along with a simultaneous decrease in the levels of H₂O₂. In the presence of H₂O₂, a significant rise in components of the pentose pathway and specific amino acids such as methionine and serine occurred, along with pathways related to an increase in antioxidant species metabolism such as ribulose 5-phosphate and glyceric acid. Conversely, fatty acid and steroid levels decrease. We found no common increase in metabolites or lipids. In contrast, eight species (succinic acid, glutamic acid, valine, 2-hydroxyisocaproic acid, alanine, indolelactic acid, proline, and lanosterol) were consumed under both stresses. These findings underscore the rapid and distinct enrichment responses in amino acids, lipids, and carbohydrates required to cope with each different environmental condition. We concluded that T. cruzi presents a flexible metabolism that rapidly adapts to variable changes in the environment.

Trypanosoma cruzi, the etiological agent of Chagas disease is a protozoan parasite that infects phagocytic and non-phagocytic mammalian cells. At early stages of infection, trypomastigotes, the infective forms of this parasite, localize in a vesicular compartment called the T. cruzi parasitophorous vacuole until the exit of parasites to the host cell cytoplasm where continue their infective cycle. Rab proteins participate in the membrane traffic's molecular machinery, functioning as central regulators of vesicle recognition and transport. In previous work, we demonstrated that endocytic Rabs are key factors of the T. cruzi infection process in non-phagocytic cells, regulating the formation and the maturation of the vacuole. In this work, we identified and characterized other molecular components of the vesicular transport pathways and their participation in the T. cruzi infection. We found that Rab9a and Rab32, two regulators of the endocytic and autophagic pathways, were actively recruited to the T. cruzi vacuoles and favored the late stages of the infective process. The recruitment was specific and dependent on T. cruzi protein synthesis. Interestingly, Rab32 association depends on the presence of Rab9a in the vacuolar membrane, while the inhibition of the cysteine-protease cruzipain, a T. cruzi virulence factor, significantly decreases both Rab9a and Rab32 association with the vacuole. In summary, this work showed for the first time that specific molecules produced and secreted by the parasite can subvert intracellular components of host cells to benefit the infection. These new data shed light on the complex map of interactions between T. cruzi and the host cell and introduce concepts that can be useful in finding new forms of intervention against this parasite in the future.

The study of host-pathogen interactions has increased considerably in recent decades. This intercellular communication has been mediated by extracellular vesicles (EVs) that play an important role during the interaction. EVs are particles of lipid bilayer and described in different types of cells, eukaryotic or prokaryotic. Depending on their biogenesis they are described as exosomes (derived from multivesicular bodies) and microvesicles (derived from the plasma membrane). The EVs carry biomolecules, including nucleic acids, lipids, and proteins that can be released or internalized by other cells in different pathways (endocytosis, macropinocytosis, phagocytosis, or membrane fusion) in the process described as uptake. The balance between biogenesis and uptake of EVs could modify physiological and pathophysiological processes of the cell. This review is focusing on the dynamic roles of release and capture of EVs during host-pathogen interaction. We also do a critical analysis of methodologies for obtaining and analyzing EVs. Finally, we draw attention to critical points to be considered in EV biogenesis and uptake studies.

Aedes aegypti females are natural vectors of important arboviruses such as dengue, zika, and yellow fever. Mosquitoes activate innate immune response signaling pathways upon infection, as a resistance mechanism to fight pathogens and limit their propagation. Despite the beneficial effects of immune activation for insect vectors, phenotypic costs ultimately affect their fitness. However, the underlying mechanisms that mediate these fitness costs remain poorly understood. Given the high energy required to mount a proper immune response, we hypothesized that systemic activation of innate immunity would impair flight muscle mitochondrial function, compromising tissue energy demand and flight activity. Here, we investigated the dynamic effects of activation of innate immunity by intra-thoracic zymosan injection on A. aegypti flight muscle mitochondrial metabolism. Zymosan injection significantly increased defensin A expression in fat bodies in a time-dependent manner that compromised flight activity. Although oxidant levels in flight muscle were hardly altered, ATP-linked respiratory rates driven by mitochondrial pyruvate+proline oxidation were significantly reduced at 24 h upon zymosan injection. Oxidative phosphorylation coupling was preserved regardless of innate immune response activation along 24 h. Importantly, rotenone-sensitive respiration and complex I-III activity were specifically reduced 24 h upon zymosan injection. Also, loss of complex I activity compromised ATP-linked and maximal respiratory rates mediated by mitochondrial proline oxidation. Finally, the magnitude of innate immune response activation negatively correlated with respiratory rates, regardless of the metabolic states. Collectively, we demonstrate that activation of innate immunity is strongly associated with reduced flight muscle complex I activity with direct consequences to mitochondrial proline oxidation and flight activity. Remarkably, our results indicate a trade-off between dispersal and immunity exists in an insect vector, underscoring the potential consequences of disrupted flight muscle mitochondrial energy metabolism to arbovirus transmission.

Bacteria adapt the biosynthesis of their envelopes to specific growth conditions and prevailing stress factors. Peptidoglycan (PG) is the major component of the cell wall in Gram-positive bacteria, where PASTA kinases play a central role in PG biosynthesis regulation. Despite their importance for growth, cell division and antibiotic resistance, the mechanisms of PASTA kinase activation are not fully understood. ReoM, a recently discovered cytosolic phosphoprotein, is one of the main substrates of the PASTA kinase PrkA in the Gram-positive human pathogen Listeria monocytogenes. Depending on its phosphorylation, ReoM controls proteolytic stability of MurA, the first enzyme in the PG biosynthesis pathway. The late cell division protein GpsB has been implicated in PASTA kinase signalling. Consistently, we show that L. monocytogenes prkA and gpsB mutants phenocopied each other. Analysis of in vivo ReoM phosphorylation confirmed GpsB as an activator of PrkA leading to the description of structural features in GpsB that are important for kinase activation. We further show that ReoM phosphorylation is growth phase-dependent and that this kinetic is reliant on the protein phosphatase PrpC. ReoM phosphorylation was inhibited in mutants with defects in MurA degradation, leading to the discovery that MurA overexpression prevented ReoM phosphorylation. Overexpressed MurA must be able to bind its substrates and interact with ReoM to exert this effect, but the extracellular PASTA domains of PrkA or MurJ flippases were not required. Our results indicate that intracellular signals control ReoM phosphorylation and extend current models describing the mechanisms of PASTA kinase activation.

Sinorhizobium meliloti senses nutrients and compounds exuded from alfalfa host roots and coordinates an excitation, termination, and adaptation pathway during chemotaxis. We investigated the role of the novel S. meliloti chemotaxis protein CheT. While CheT and the Escherichia coli phosphatase CheZ share little sequence homology, CheT is predicted to possess an α-helix with a DXXXQ phosphatase motif. Phosphorylation assays demonstrated that CheT dephosphorylates the phosphate-sink response regulator, CheY1~P by enhancing its decay two-fold but does not affect the motor response regulator CheY2~P. Isothermal Titration Calorimetry (ITC) experiments revealed that CheT binds to a phosphomimic of CheY1~P with a K_D of 2.9 μM, which is 25-fold stronger than its binding to CheY1. Dissimilar chemotaxis phenotypes of the ΔcheT mutant and cheT DXXXQ phosphatase mutants led to the hypothesis that CheT exerts additional function(s). A screen for potential binding partners of CheT revealed that it forms a complex with the methyltransferase CheR. ITC experiments confirmed CheT/CheR binding with a K_D of 19 μM, and a SEC-MALS analysis determined a 1:1 and 2:1 CheT/CheR complex formation. Although they did not affect each other's enzymatic activity, CheT binding to CheY1~P and CheR may serve as a link between signal termination and sensory adaptation.

Many chemoreceptors contain a C-terminal pentapeptide at the end of a linker. In Escherichia coli, this pentapeptide forms a high-affinity binding site for CheR and phosphorylated CheB, and its removal interferes with chemoreceptor adaptation. Analysis of chemoreceptors revealed significant variation in their pentapeptide sequences, and bacteria often possess multiple chemoreceptors with differing pentapeptides. To assess whether this sequence variation alters CheR affinity and chemotaxis, we used Pectobacterium atrosepticum SCRI1043 as a model. SCRI1043 has 36 chemoreceptors, with 19 of them containing a C-terminal pentapeptide. We show that the affinity of CheR for the different pentapeptides varies up to 11-fold (K_D 90 nM to 1 μM). Pentapeptides with the highest and lowest affinities differ only in a single amino acid. Deletion of the cheR gene abolishes chemotaxis. The replacement of the pentapeptide in the PacC chemoreceptor with those of the highest and lowest affinities significantly reduced chemotaxis to its cognate chemoeffector, L-Asp. Altering the PacC pentapeptide also reduced chemotaxis to L-Ser, but not to nitrate, which are responses mediated by the nontethered PacB and PacN chemoreceptors, respectively. Changes in the pentapeptide sequence thus modulate the response of the cognate receptor and that of another chemoreceptor.

Many bacteria possess proteasomes and a tagging system that is functionally analogous to the ubiquitin system. In this system, Pup, the tagging protein, marks protein targets for proteasomal degradation. Despite the analogy to the ubiquitin system, where the ubiquitin tag is recycled, it remained unclear whether Pup is similarly recycled, given how the bacterial proteasome does not include a depupylase. We previously showed in vitro that as Pup lacks effective proteasome degradation sites, it is released from the proteasome following target degradation, remaining conjugated to a degradation fragment that can be later depupylated. Here, we tested this model in Mycobacterium smegmatis, using a Pup mutant that is effectively degraded by the proteasome. Our findings indicate that Pup recycling not only occurs in vivo but is also essential to maintain normal pupylome levels and to support bacterial survival under starvation conditions. Accordingly, Pup recycling is an essential process in the mycobacterial Pup-proteasome system.