Intracellular pathogens have developed different mechanisms which enable their survival and replication within the host cells. Some survive and replicate within a membrane-bound vacuole modified by the bacteria to support microbial growth (e.g. Salmonella enterica serovar Typhimurium), whereas others escape from the vacuole into the host cell cytosol, where they proliferate (e.g. Listeria monocytogenes). In this study a Salmonella strain carrying a mutation in sifA which is released from the vacuole was used to analyse Salmonella survival and replication within the cytosol of several cell lines. It was found that Salmonella replicates within the cytosol of epithelial cells at a higher rate than that achieved when replicating within the vacuole, but is defective for replication in the cytosol of fibroblasts or macrophages. Using an aroC purD double mutant strain which does not replicate within host cells, it was shown that Salmonella encounters a killing activity within the cytosol of macrophages. Furthermore, in vitro experiments using cytosol extracted from either infected or uninfected macrophages suggested that this activity is activated upon Salmonella infection.
27Brucella species are facultative intracellular Gram-negative bacteria relevant to animal 28 and human health. Their ability to establish an intracellular niche and subvert host cell 29 pathways to their advantage depends on the delivery of bacterial effector proteins 30 through a type IV secretion system. Brucella Toll/Interleukin-1 Receptor (TIR)-domain-31 containing proteins BtpA (also known as TcpB) and BtpB are among such effectors. 32Although divergent in primary sequence, they interfere with Toll-like receptor (TLR) 33 signaling to inhibit the innate immune responses. However, the molecular mechanisms 34 implicated still remain unclear. To gain insight into the functions of BtpA and BtpB, we 35 expressed them in the budding yeast Saccharomyces cerevisiae as a eukaryotic cell 36 model. We found that both effectors were cytotoxic and that their respective TIR 37 domains were necessary and sufficient for yeast growth inhibition. Growth arrest was 38 concomitant with actin depolymerization, endocytic block and a general decrease in 39 kinase activity in the cell, suggesting a failure in energetic metabolism. Indeed, levels of 40 ATP and NAD + were low in yeast cells expressing BtpA and BtpB TIR domains, 41 consistent with the recently described enzymatic activity of some TIR domains as 42 NAD + hydrolases. In human epithelial cells, both BtpA and BtpB expression reduced 43 intracellular total NAD levels. In infected cells, both BtpA and BtpB contributed to 44 reduction of total NAD, indicating that their NAD + hydrolase functions are active 45 intracellularly during infection. Overall, combining the yeast model together with 46 mammalian cells and infection studies our results show that BtpA and BtpB modulate 47 AUTHOR SUMMARY 51Brucella is a genus of zoonotic bacteria that cause severe disease in a variety of 52 mammals, ranging from farm animals (as bovines, swine and ovine) to marine 53 mammals. Transmission to humans, often by ingestion of non-treated dairy products, 54 leads to serious systemic infection. Brucella abortus invades host cells and replicates 55intracellularly. Such behavior relies on the injection of bacterial proteins into the host 56 cytoplasm via specialized secretion systems. Our work focuses on the study of two of 57 these factors, BtpA and BtpB, previously described to contain Toll/Interleukin-1 58Receptor (TIR)-domains that modulate innate immunity. We use here two biological 59 models: the yeast Saccharomyces cerevisiae and human cell lines. We found that the 60 TIR domains of both Brucella proteins were necessary and sufficient to collapse energy 61 metabolism in yeast by depleting ATP and NAD + . This result was translatable to higher 62 cells and consistent with the recently described NADase activity of some TIR domains 63 both in mammalian and bacterial proteins. Importantly, we demonstrate that Brucella 64 down-regulates total NAD levels in host cells by using both BtpA and BtpB effectors. 65Our results show that NAD + is targeted by Brucella during infection, which may 66 cons...
The spread of antibiotic resistant Acinetobacter baumannii poses a significant threat to public health worldwide. This nosocomial bacterial pathogen can be associated with life-threatening infections, particularly in intensive care units. A. baumannii is mainly described as an extracellular pathogen with restricted survival within cells. This study shows that a subset of A. baumannii clinical isolates extensively multiply within non-phagocytic immortalized and primary cells, without the induction of apoptosis, and with bacterial clusters visible up to 48 hours after infection. This phenotype was observed for the A. baumannii C4 strain associated with high mortality in a hospital outbreak, and the A. baumannii ABC141 strain which wasn't isolated from an infection site but was found to be hyperinvasive. Intracellular multiplication of these A. baumannii strains occurred within spacious single membrane-bound vacuoles, labeled with the lysosomal associate membrane protein (LAMP1). However, these compartments excluded lysotracker, an indicator of acidic pH, suggesting that A. baumannii can divert its trafficking away from the lysosomal degradative pathway. These compartments were also devoid of autophagy features. A high-content microscopy screen of 43 additional A. baumannii clinical strains highlighted various phenotypes: (1) the majority of strains remained extracellular, (2) a significant proportion was capable of invasion and limited persistence, and (3) two strains efficiently multiplied within LAMP1-positive vacuoles, one of which was also hyperinvasive. These data identify an intracellular niche for specific A. baumannii clinical strains that enables extensive multiplication in an environment protected from host immune responses and out of reach from many antibiotics.
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