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Inorganic polyphosphate is essential for long-term survival and virulence factors in Shigella and Salmonella spp.

ABSTRACT: The importance of inorganic polyphosphate (poly P) and poly P kinase (PPK), the enzyme principally responsible for its synthesis, has been established previously for stationary-phase survival of Escherichia coli and virulence in Pseudomonas aeruginosa. The gene (ppk) that encodes PPK is highly conserved among many bacterial pathogens, including Shigella and Salmonella spp. In view of the phylogenetic similarity of the enteropathogens and the frequency with which virulence factors are expressed in stationary phase, the ppk gene of pathogenic Shigella flexneri, Salmonella enterica serovar Dublin, and Salmonella enterica serovar typhimurium have been cloned and deleted. In some of these mutants lacking ppk, the phenotypes included features indicative of decreased virulence such as: (i) growth defects, (ii) defective responses to stress and starvation, (iii) loss of viability, (iv) polymyxin sensitivity, (v) intolerance to acid and heat, and (vi) diminished invasiveness in epithelial cells. Thus PPK may prove, as it has with P. aeruginosa, to be an attractive target for antibiotics, with low toxicity because PPK is not found in higher eukaryotes.

SUBMITTER: Kim KS

PROVIDER: S-EPMC124319 | biostudies-literature | 2002 May

REPOSITORIES: biostudies-literature

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Inorganic polyphosphate is essential for long-term survival and virulence factors in Shigella and Salmonella spp.

Kim Kwang-Seo KS Rao Narayana N NN Fraley Cresson D CD Kornberg Arthur A

Proceedings of the National Academy of Sciences of the United States of America 20020501 11

The importance of inorganic polyphosphate (poly P) and poly P kinase (PPK), the enzyme principally responsible for its synthesis, has been established previously for stationary-phase survival of Escherichia coli and virulence in Pseudomonas aeruginosa. The gene (ppk) that encodes PPK is highly conserved among many bacterial pathogens, including Shigella and Salmonella spp. In view of the phylogenetic similarity of the enteropathogens and the frequency with which virulence factors are expressed i ...[more]

PMID: 12032342

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Project description:Inorganic polyphosphate (polyP) deficiency in enteric bacterial pathogens reduces their ability to invade and establish systemic infections in different hosts. For instance, inactivation of the polyP kinase gene (ppk) encoding the enzyme responsible for polyP biosynthesis reduces invasiveness and intracellular survival of Salmonella enterica serovar Typhimurium (S. Typhimurium) in epithelial cells and macrophages in vitro. In addition, the virulence in vivo of a S. Typhimurium ?ppk mutant is significantly reduced in a murine infection model. In spite of these observations, the role played by polyP during the Salmonella-host interaction is not well understood. The social amoeba Dictyostelium discoideum has proven to be a useful model for studying relevant aspects of the host-pathogen interaction. In fact, many intracellular pathogens can survive within D. discoideum cells using molecular mechanisms also required to survive within macrophages. Recently, we established that S. Typhimurium is able to survive intracellularly in D. discoideum and identified relevant genes linked to virulence that are crucial for this process. The aim of this study was to determine the effect of a polyP deficiency in S. Typhimurium during its interaction with D. discoideum. To do this, we evaluated the intracellular survival of wild-type and ?ppk strains of S. Typhimurium in D. discoideum and the ability of these strains to delay the social development of the amoeba. In contrast to the wild-type strain, the ?ppk mutant was unable to survive intracellularly in D. discoideum and enabled the social development of the amoeba. Both phenotypes were complemented using a plasmid carrying a copy of the ppk gene. Next, we simultaneously evaluated the proteomic response of both S. Typhimurium and D. discoideum during host-pathogen interaction via global proteomic profiling. The analysis of our results allowed the identification of novel molecular signatures that give insight into Salmonella-Dictyostelium interaction. Altogether, our results indicate that inorganic polyP is essential for S. Typhimurium virulence and survival in D. discoideum. In addition, we have validated the use of global proteomic analyses to simultaneously evaluate the host-pathogen interaction of S. Typhimurium and D. discoideum. Furthermore, our infection assays using these organisms can be exploited to screen for novel anti-virulence molecules targeting inorganic polyP biosynthesis.

Project description:The Mycobacterium tuberculosis gene Rv3232c/MT3329 (ppk2) encodes a class II polyphosphate kinase, which hydrolyzes inorganic polyphosphate (poly P) to synthesize GTP. We assessed the role of ppk2 in M. tuberculosis poly P regulation, antibiotic tolerance, and virulence. A ppk2-deficient mutant (ppk2::Tn) and its isogenic wild-type (WT) and complemented (Comp) strains were studied. For each strain, the intrabacillary poly P content, MIC of isoniazid, and growth kinetics during infection of J774 macrophages were determined. Multiplex immunobead assays were used to evaluate cytokines elaborated during macrophage infection. The requirement of ppk2 for M. tuberculosis virulence was assessed in the murine model. The ppk2::Tn mutant was found to have significantly increased poly P content and a 4-fold increase in the MIC of isoniazid relative to the WT and Comp strains. The ppk2::Tn mutant showed reduced survival at day 7 in activated and naive J774 macrophages relative to the WT. Naive ppk2::Tn mutant-infected macrophages showed increased expression of interleukin 2 (IL-2), IL-9, IL-10, IL-12p70, and gamma interferon (IFN-?) relative to WT-infected macrophages. The ppk2::Tn mutant exhibited significantly lower lung CFU during acute murine infection compared to the control groups. ppk2 is required for control of intrabacillary poly P levels and optimal M. tuberculosis growth and survival in macrophages and mouse lungs. IMPORTANCE Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a highly successful human pathogen because it has developed mechanisms to multiply and survive in the lungs by circumventing the immune system. Identification of virulence factors responsible for M. tuberculosis growth and persistence in host tissues may assist in the development of novel strategies to treat TB. In this study, we found that the mycobacterial enzyme polyphosphate kinase 2 (PPK2) is required for controlling intracellular levels of important regulatory molecules and for maintaining susceptibility to the first-line anti-TB drug isoniazid. In addition, PPK2 was found to be required for M. tuberculosis growth in the lungs of mice, at least in part by suppressing the expression of certain key cytokines and chemokines by inactivated lung macrophages.

Project description:Leprosy is a curable neglected disease of humans caused by Mycobacterium leprae that affects the skin and peripheral nerves and manifests clinically in various forms ranging from self-resolving, tuberculoid leprosy to lepromatous leprosy having significant pathology with ensuing disfiguration disability and social stigma. Despite the global success of multi-drug therapy (MDT), incidences of clinical leprosy have been observed in individuals with no apparent exposure to other cases, suggestive of possible non-human sources of the bacteria. In this study we show that common free-living amoebae (FLA) can phagocytose M. leprae, and allow the bacillus to remain viable for up to 8 months within amoebic cysts. Viable bacilli were extracted from separate encysted cocultures comprising three common Acanthamoeba spp.: A. lenticulata, A. castellanii, and A. polyphaga and two strains of Hartmannella vermiformis. Trophozoites of these common FLA take up M. leprae by phagocytosis. M. leprae from infected trophozoites induced to encyst for long-term storage of the bacilli emerged viable by assessment of membrane integrity. The majority (80%) of mice that were injected with bacilli extracted from 35 day cocultures of encysted/excysted A. castellanii and A. polyphaga showed lesion development that was similar to mice challenged with fresh M. leprae from passage mice albeit at a slower initial rate. Mice challenged with coculture-extracted bacilli showed evidence of acid-fast bacteria and positive PCR signal for M. leprae. These data support the conclusion that M. leprae can remain viable long-term in environmentally ubiquitous FLA and retain virulence as assessed in the nu/nu mouse model. Additionally, this work supports the idea that M. leprae might be sustained in the environment between hosts in FLA and such residence in FLA may provide a macrophage-like niche contributing to the higher-than-expected rate of leprosy transmission despite a significant decrease in human reservoirs due to MDT.

Project description:The emergence of hypervirulent Klebsiella pneumoniae (hvKP) strains poses a significant threat to public health due to their high mortality rates and propensity to cause severe community-acquired infections in otherwise healthy individuals. The ability of hvKP to form biofilms and produce a protective capsule contributes to its enhanced virulence and is a significant challenge to effective antibiotic treatment. Therefore, understanding the molecular mechanisms underlying hvKP virulence and biofilm formation is crucial for developing new therapeutic strategies. Polyphosphate Kinase 1 (PPK1) is an enzyme responsible for inorganic polyphosphate synthesis and plays a vital role in regulating various physiological processes in bacteria. In this study, we investigated the impact of polyP metabolism on the biofilm and capsule formation and virulence traits in hvKP using Dictyostelium discoideum amoeba as a model host. We found that the PPK1 null-mutant was impaired in biofilm and capsule formation and showed attenuated virulence in D. discoideum compared to the wild-type strain. We performed a shotgun proteomic analysis of the PPK1 mutant and wild-type strain to gain further insight into the underlying molecular mechanism. The results revealed that the PPK1 mutant had a differential expression of proteins (DEP) involved in capsule synthesis (Wzi - Ugd), biofilm formation (MrkC-D-H), synthesis of the colibactin genotoxin precursor (ClbB), as well as proteins associated with the synthesis and modification of lipid A (ArnB -LpxC - PagP). These proteomic findings corroborate the phenotypic observations and indicate that the PPK1 mutation is associated with impaired biofilm and capsule formation and attenuated virulence in hypervirulent K. pneumoniae. Overall, our study highlights the importance of polyP synthesis in regulating extracellular biomolecules and virulence in K. pneumoniae and provides insights into potential therapeutic targets for treating K. pneumoniae infections.

Dataset Information

Inorganic polyphosphate is essential for long-term survival and virulence factors in Shigella and Salmonella spp.

Publications

Inorganic polyphosphate is essential for long-term survival and virulence factors in Shigella and Salmonella spp.

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