Molecular Characterization of SehB, a Type II Antitoxin of Salmonella enterica Serotype Typhimurium: Amino Acid Residues Involved in DNA-Binding, Homodimerization, Toxin Interaction, and Virulence.

Salmonella enterica serotype Typhimurium is a bacterium that causes gastroenteritis and diarrhea in humans. The genome of S. Typhimurium codes for diverse virulence factors, among which are the toxin-antitoxin (TA) systems. SehAB is a type II TA, where SehA is the toxin and SehB is the antitoxin. It was previously reported that the absence of the SehB antitoxin affects the growth of S. Typhimurium. In addition, the SehB antitoxin can interact directly with the SehA toxin neutralizing its toxic effect as well as repressing its own expression. We identified conserved residues on SehB homologous proteins. Point mutations were introduced at both N- and C-terminal of SehB antitoxin to analyze the effect of these changes on its transcription repressor function, on its ability to form homodimers and on the virulence of S. Typhimurium. All changes in amino acid residues at both the N- and C-terminal affected the repressor function of SehB antitoxin and they were required for DNA-binding activity. Mutations in the amino acid residues at the N-terminal showed a lower capacity for homodimer formation of the SehB protein. However, none of the SehB point mutants were affected in the interaction with the SehA toxin. In terms of virulence, the eight single-amino acid mutations were attenuated for virulence in the mouse model. In agreement with our results, the eight amino acid residues of SehB antitoxin were required for its repressor activity, affecting both homodimerization and DNA-binding activity, supporting the notion that both activities of SehB antitoxin are required to confer virulence to Salmonella enterica.

The infectious intracellular lifestyle of Salmonella enterica relies on the adaptation to nutritional conditions within the Salmonella-containing vacuole.

Salmonella enterica serovar Typhimurium (S. Typhimurium) is a Gram-negative pathogen that causes various host-specific diseases. During their life cycle, Salmonellae survive frequent exposures to a variety of environmental stresses, e.g. carbon-source starvation. The virulence of this pathogen relies on its ability to establish a replicative niche, named Salmonella-containing vacuole, inside host cells. However, the microenvironment of the SCV and the bacterial metabolic pathways required during infection are largely undefined. In this work we developed different biological probes whose expression is modulated by the environment and the physiological state of the bacterium. We constructed transcriptional reporters by fusing promoter regions to the gfpmut3a gene to monitor the expression profile of genes involved in glucose utilization and lipid catabolism. The induction of these probes by a specific metabolic change was first tested in vitro, and then during different conditions of infection in macrophages. We were able to determine that Entner-Doudoroff is the main metabolic pathway utilized by Salmonella during infection in mouse macrophages. Furthermore, we found sub-populations of bacteria expressing genes involved in pathways for the utilization of different sources of carbon. These populations are modified in presence of different metabolizable substrates, suggesting the coexistence of Salmonella with diverse metabolic states during the infection.

Activation of Rho and Rab GTPases dissociates Brucella abortus internalization from intracellular trafficking.

Brucella abortus is an intracellular pathogen that relies on unconventional virulence factors to infect hosts. In non-professional phagocytes, Rho GTPases-activation by the Escherichia coli cytotoxic necrotizing factor (CNF) promoted massive Brucella entrance by membrane ruffling, a mechanism that differs from the common mode of entrance used by this bacterium in non-treated cells. Cytotoxic necrotizing factor treatment, however, did not alter the intracellular route followed by the wild type or non-virulent defined mutants. In contrast, expression of a constitutively active Rab5Q79L GTPase did not alter cell-invasion by Brucella but hampered its ability to reach the endoplasmic reticulum. The CNF-induced Brucella super-infection did not reduce the ability of host cells to synthesize DNA and progress through the cell cycle. Furthermore, CNF-treatment increased the isolation of Brucella-containing compartments by a factor of 15. These results demonstrate that in non-professional phagocytic cells, Brucella manipulates two different sets of GTPases during its biogenesis, being internalization and intracellular trafficking two consecutive but independent processes. Besides, CNF-induced super-infection demonstrates that Brucella does not interfere with crucial cellular processes and has shown its potential as tool to characterize the intracellular compartments occupied by this bacterium.