guanosine-pentaphosphate and Disease-Models--Animal

In order to persist, bacteria need to adjust their physiological state in response to external and internal cues. External stimuli are often referred to as stressors. The stringent response, mediated by the alarmone (p)ppGpp, is central to the stress response in many bacteria; yet, there is limited knowledge regarding the role of (p)ppGpp signaling in bacteria belonging to the phylum Bacteroidetes. Like its counterparts in the gut (e.g., Bacteroides thetaiotaomicron and Bacteroides fragilis), Porphyromonas gingivalis persists in close association with its human host. Given the potential for numerous perturbations in the oral cavity, and the fact that P. gingivalis can enter and replicate within host cells, we hypothesized that (p)ppGpp is a key signaling molecule for stress adaptation and persistence. Here, we show that accumulation of ppGpp in P. gingivalis is governed by two homologous enzymes, designated Rel, and RshB, and that ppGpp signaling affects growth rate, survival, biofilm formation, production of outer membrane vesicles, and expression of genes encoding type IX secretion structural and cargo proteins. Overall, our findings provide a potential mechanism by which biofilm formation and virulence of P. gingivalis are integrated via ppGpp signaling, a regulatory mechanism central to bacterial survival in dynamic environments.

Topics: Animals; Bacterial Proteins; Bacteroidaceae Infections; Biofilms; Disease Models, Animal; Gene Expression Regulation, Bacterial; Guanosine Pentaphosphate; Lepidoptera; Porphyromonas gingivalis; Signal Transduction; Stress, Physiological; Survival Analysis; Virulence

Topics: Animals; Bacterial Proteins; Cell Survival; Disease Models, Animal; Gram-Negative Bacteria; Gram-Positive Bacteria; Guanosine Pentaphosphate; Guanosine Tetraphosphate; Immunity, Innate; Ligases; Mice; Phagosomes; Pyrophosphatases; Salmonella; Transcription Factor RelA; Virulence

How Salmonella enterica serovar Typhi (S. Typhi), an important human pathogen, survives the stressful microenvironments inside the gastrointestinal tract and within macrophages remains poorly understood. We report here that S. Typhi has a bonafide stringent response (SR) system, which is mediated by (p)ppGpp and regulates multiple virulence-associated traits and the pathogenicity of the S. Typhi Ty2 strain. In an iron overload mouse model of S. Typhi infection, the (p)ppGpp

Topics: Animals; Bacterial Proteins; Caco-2 Cells; Disease Models, Animal; Gene Expression Regulation, Bacterial; GTP Pyrophosphokinase; Guanosine Pentaphosphate; Host-Pathogen Interactions; HT29 Cells; Humans; Iron Overload; Liver; Mice; Mice, Inbred BALB C; Mice, Knockout; Polysaccharides, Bacterial; Pyrophosphatases; RAW 264.7 Cells; Salmonella typhi; Signal Transduction; Spleen; Survival Analysis; THP-1 Cells; Typhoid Fever; Virulence

Topics: Animals; Bacterial Proteins; Biofilms; Cells, Cultured; Disease Models, Animal; Endocarditis, Bacterial; Endothelial Cells; Enterococcus faecalis; Gelatinases; Gene Deletion; Gene Expression Regulation, Bacterial; Guanosine Pentaphosphate; Humans; Ligases; Rabbits; Swine; Virulence

During infection, bacteria might generate adaptive responses to facilitate their survival and colonization in the host environment. The alarmone guanosine 5'-triphosphate-3'-diphosphate (ppGpp), the levels of which are regulated by the RelA and SpoT enzymes, plays a critical role in mediating bacterial adaptive responses and virulence. However, the mechanism by which ppGpp regulates virulence-associated traits in Pseudomonas aeruginosa is poorly understood. To investigate the regulatory role of ppGpp, the ppGpp-deficient strain ΔRS (relA and spoT gene double mutant) and the complemented strain ΔRS(++) (complemented with relA and spoT genes) were constructed. Herein, we reported that the ΔRS strain showed decreased cytotoxicity towards A549 human alveolar adenocarcinoma cell lines and led to reduced mortality, lung edema and inflammatory cell infiltration in a mouse model of acute pneumonia compared to wild-type PAO1 and the complemented strain ΔRS(++). Subsequent analyses demonstrated that the ΔRS strain displayed reduced T3SS expression, decreased levels of elastase activity, pyocyanin, pyoverdin and alginate, and inhibited swarming and biofilm formation compared to PAO1 and the complemented strain ΔRS(++). In addition, the results demonstrate that ppGpp-mediated regulation of T3SS, virulence factor production, and swarming occurs in a quinolone quorum-sensing system-dependent manner. Taken together, these results suggest that ppGpp is required for virulence regulation in P. aeruginosa, providing new clues for the development of interference strategies against bacterial infection.

Topics: A549 Cells; Animals; Bacterial Proteins; Biofilms; Disease Models, Animal; Gene Deletion; Guanosine Pentaphosphate; Humans; Male; Mice; Microbial Viability; Phenotype; Pneumonia, Bacterial; Pseudomonas aeruginosa; Pseudomonas Infections; Quorum Sensing; Type III Secretion Systems; Virulence; Virulence Factors

In Mycobacterium tuberculosis, the stringent response to amino acid starvation is mediated by the M. tuberculosis Rel (RelMtb) enzyme, which transfers a pyrophosphate from ATP to GDP or GTP to synthesize ppGpp and pppGpp, respectively. (p)ppGpp then influences numerous metabolic processes. RelMtb also encodes a second, distinct catalytic domain that hydrolyzes (p)ppGpp into pyrophosphate and GDP or GTP. RelMtb is required for chronic M. tuberculosis infection in mice; however, it is unknown which catalytic activity of RelMtb mediates pathogenesis and whether (p)ppGpp itself is necessary. In order to individually investigate the roles of (p)ppGpp synthesis and hydrolysis during M. tuberculosis pathogenesis, we generated RelMtb point mutants that were either synthetase dead (RelMtb(H344Y)) or hydrolase dead (RelMtb(H80A)). M. tuberculosis strains expressing the synthetase-dead RelMtb(H344Y) mutant did not persist in mice, demonstrating that the RelMtb (p)ppGpp synthetase activity is required for maintaining bacterial titers during chronic infection. Deletion of a second predicted (p)ppGpp synthetase had no effect on pathogenesis, demonstrating that RelMtb was the major contributor to (p)ppGpp production during infection. Interestingly, expression of an allele encoding the hydrolase-dead RelMtb mutant, RelMtb(H80A), that is incapable of hydrolyzing (p)ppGpp but still able to synthesize (p)ppGpp decreased the growth rate of M. tuberculosis and changed the colony morphology of the bacteria. In addition, RelMtb(H80A) expression during acute or chronic M. tuberculosis infection in mice was lethal to the infecting bacteria. These findings highlight a distinct role for RelMtb-mediated (p)ppGpp hydrolysis that is essential for M. tuberculosis pathogenesis.

Topics: Animals; Bacteria; Disease Models, Animal; Guanosine Pentaphosphate; Guanosine Tetraphosphate; Ligases; Mice; Mutant Proteins; Mutation, Missense; Mycobacterium tuberculosis; Point Mutation; Survival Analysis; Tuberculosis; Virulence Factors

The stringent response is a regulatory system that allows bacteria to sense and adapt to nutrient-poor environments. The central mediator of the stringent response is the molecule guanosine 3',5'-bispyrophosphate (ppGpp), which is synthesized by the enzymes RelA and SpoT and which is also degraded by SpoT. Our laboratory previously demonstrated that a relA mutant of Pseudomonas aeruginosa, the principal cause of lung infections in cystic fibrosis patients, was attenuated in virulence in a Drosophila melanogaster feeding model of infection. In this study, we examined the role of spoT in P. aeruginosa virulence. We generated an insertion mutation in spoT within the previously constructed relA mutant, thereby producing a ppGpp-devoid strain. The relA spoT double mutant was unable to establish a chronic infection in D. melanogaster and was also avirulent in the rat lung agar bead model of infection, a model in which the relA mutant is fully virulent. Synthesis of the virulence determinants pyocyanin, elastase, protease, and siderophores was impaired in the relA spoT double mutant. This mutant was also defective in swarming and twitching, but not in swimming motility. The relA spoT mutant and, to a lesser extent, the relA mutant were less able to withstand stresses such as heat shock and oxidative stress than the wild-type strain PAO1, which may partially account for the inability of the relA spoT mutant to successfully colonize the rat lung. Our results indicate that the stringent response, and SpoT in particular, is a crucial regulator of virulence processes in P. aeruginosa.

Topics: Agar; Animals; Bacterial Load; Bacterial Proteins; Disease Models, Animal; Drosophila melanogaster; Feeding Behavior; Gene Expression Regulation, Bacterial; Guanosine Pentaphosphate; Heat-Shock Response; Humans; Ligases; Lung; Mutation; Pseudomonas aeruginosa; Pseudomonas Infections; Rats; Virulence