Role of the Neisseria meningitidis PTS component HPr in the correlation between carbon metabolism and virulence – MeniViruCarb

The PTS component HPr seems to play a major role in the correlation between carbon metabolism and virulence in N. meningitidis. Its inactivation leads to lower capsule production and enhanced cell adhesion. In addition, expression of the crgA gene, which codes for the regulator of the pili and capsule operons, is elevated in the ptsH mutant. HPr has been shown to interact with CrgA and probably affects the binding activities of the transcription regulator for its DNA target sites. We want to study, which HPr forms (unphosphorylated, phosphorylated at His-15 or at Ser-46, or doubly phosphorylated HPr) are responsible for the observed effects. For this purpose we will construct a series of mutants synthesizing either unphosphorylatable HPr or HPr with potentially phosphomimetic replacements at the phosphorylatation sites. Specific mutants affected in the genes encoding the EIIAFru and the EIIAMan components of the N. meningitdis PTS will also be constructed. The effect of these mutations on N. meningitidis virulence and cell adhesion will be tested in an animal model (mice expressing human transferrin) and in cell culture. After intraperitoneal challenge, levels of bacteremia will be assessed and the inflammatory response will be analyzed.
In order to indentify the detailed molecular mechanism of the signal transduction pathway connecting carbon metabolism and virulence in N. meningitidis we will also carry out a transcriptome analysis with the ptsH mutant. Genes exhibiting a differential expression in wild-type and ptsH mutant will be studied in detail by constructing corresponding mutants and theencoded proteins. In addition, we will try to identify interaction partners of phosphorylated and unphosphorylated PTS components by carrying out SPINE and co-immunoprecipitation experiments.

We constructed a Neisseria meningitidis mutant carrying a deletion of the ptsH gene. In order to complement this mutant with different ptsH alleles we constructed the dsired alleles (His15Ala, His15Asp, Ser46Ala, Ser46Asp et des doubles mutations) and inserted them in a plasmid allowing the insertion into the N. meningitidis genome. The effect of these mutations on CrgA activity, bacterial survival in an animal model and in cell cultures as well as on apopotosis is presently studued. In a second step, these mutants will also be used to cary out trasncriptome analyses.
We also tested a possible interaction between HPr and CrgA by carrying out yeast two hybrid experiments in collaboration with the group of Philippe NOIROT, INRA in Jouy en Josas. The wild-type proteins did not show an interaction. We are now testing interactions of various mutant HPr’s (His15Asp, Ser46Asp et la protéine doublement mutée) with CrgA.
We also impoved the purification conditions for HPr and CrgA, which have the tendency to form inclusion bodies., in order to carry out in vitro interaction assays with these two proteins as well as to study interactions of CrgA or the CrgA/HPr complex with the cognate operator site (CrgA box with and without a CREN).

Mutations of the genes encoding components of the incomplete menigococcal phosphotransferase system affect capsule production. This bacterial cell surface component plays an important role in cell adhesion. Because N. meningitidis represents a major threat to human health, understanding the fundamental processes connecting carbon metabolism and virulence is expected to allow a more efficient and a better targeted treatment with already existing drugs and/or to provide the basis for the development of new antimicrobial agents.

We constructed a Neisseria meningitidis serogroup C mutant carrying a deletion of the ptsH gene. We also constructed numerous ptsH alleles carrying mutations leading to the replacement of the phosphorylatable amino acids in HPr with non-phosphoryltable amino acids or with amino acids mimicking une phosphorylation. Finally, we constructed several vectors for the yeast two hybrid experiemnts and plamsids used for the overexpression of the ptsH and crgA genes and the purification of the encoded proteins HPr and CrgA, respectively.

Neisseria meningitidis is an exclusively human pathogen, which represents a major threat to health. It is commensal of the nasopharynx with 10 to 15% of the population in Europe being asymptotic carriers. Upon specific signals, which have not yet been identified, N. menigitidis crosses the epithelial cell layer and enters into the bloodstream. It is possible that carbon sources play a critical role in the change from commensal to virulent state. Indeed, results from an ongoing collaboration suggest that components of the PEP:glycose phosphotransferase system (PTS), a transport system for sugars and sugar derivatives, play a role in virulence regulation. Nevertheless, N. meningitidis possesses an incomplete PTS unable to transport any sugar. Glucose and maltose are the only carbohydrates taken up by this organism via non-PTS transporters.
The PTS component HPr seems to play a major role in the correlation between carbon metabolism and virulence in N. meningitidis. Its inactivation leads to lower capsule production and enhanced cell adhesion. In addition, expression of the crgA gene, which codes for the regulator of the pili and capsule operons, is elevated in the ptsH mutant. HPr has been shown to interact with CrgA and probably affects the binding activities of the transcription regulator for its DNA target sites. We want to study, which HPr forms (unphosphorylated, phosphorylated at His-15 or at Ser-46, or doubly phosphorylated HPr) are responsible for the observed effects. For this purpose we will construct a series of mutants synthesizing either unphosphorylatable HPr or HPr with potentially phosphomimetic replacements at the phosphorylatation sites. Specific mutants affected in the genes encoding the EIIAFru and the EIIAMan components of the N. meningitdis PTS will also be constructed. The effect of these mutations on N. meningitidis virulence and cell adhesion will be tested in an animal model (mice expressing human transferrin) and in cell culture. After intraperitoneal challenge, levels of bacteremia will be assessed and the inflammatory response will be analyzed.
In order to indentify the detailed molecular mechanism of the signal transduction pathway connecting carbon metabolism and virulence in N. meningitidis we will also carry out a transcriptome analysis with the ptsH mutant. Genes exhibiting a differential expression in wild-type and ptsH mutant will be studied in detail by constructing corresponding mutants and theencoded proteins. In addition, we will try to identify interaction partners of phosphorylated and unphosphorylated PTS components by carrying out SPINE and co-immunoprecipitation experiments