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Molecular characterization and identification of proteins regulated by Hfq in Neisseria meningitidis.

Pannekoek Y, Huis in 't Veld R, Hopman CT, Langerak AA, Speijer D, van der Ende A - FEMS Microbiol. Lett. (2009)

Bottom Line: Using proteomics, the expression of 28 proteins was found to be significantly affected upon deletion of hfq.One of the differentially expressed proteins, GdhA, was identified as an essential virulence factor for establishment of sepsis in an animal model, studied earlier.These results show that in N. meningitidis Hfq is involved in the regulation of a variety of components contributing to the survival and establishment of meningococcal disease.

View Article: PubMed Central - PubMed

ABSTRACT
Hfq is a highly conserved pleiotropically acting prokaryotic RNA-binding protein involved in the post-transcriptional regulation of many stress-responsive genes by small RNAs. In this study, we show that Hfq of the strictly human pathogen Neisseria meningitidis is involved in the regulation of expression of components involved in general metabolic pathways, iron metabolism and virulence. A meningococcal hfq deletion strain (H44/76Deltahfq) is impaired in growth in nutrient-rich media and does not grow at all in nutrient-limiting medium. The growth defect was complemented by expression of hfq in trans. Using proteomics, the expression of 28 proteins was found to be significantly affected upon deletion of hfq. Of these, 20 proteins are involved in general metabolism, among them seven iron-responsive genes. Two proteins (PilE, TspA) are involved in adherence to human cells, a step crucial for the onset of disease. One of the differentially expressed proteins, GdhA, was identified as an essential virulence factor for establishment of sepsis in an animal model, studied earlier. These results show that in N. meningitidis Hfq is involved in the regulation of a variety of components contributing to the survival and establishment of meningococcal disease.

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Amino acid sequence alignment of Hfq proteins of Escherichia coli serotypes, Vibrio species, Neisseria meningitidis, Neisseria gonorrhoeae strains and Neisseria lactamica. The secondary structural elements of Hfq protein are shown above the alignment with the N-terminal helix α1 in cyan. The Sm1 and Sm2 motif regions are boxed. The sole conserved residue Gly34 is indicated in red. Highly conserved hydrophobic residues within the Sm1 region are indicated by a lower case h, and the two highly conserved glycine and aspartic acid residues within the Sm1 motif are indicated by G and D, respectively. The absolutely conserved glutamine of helix α1 that is important for base recognition and the highly conserved tyrosine (or phenylalanine) are indicated in green. Within the Sm2 region, the ‘Hfq Sm2 motif YKH’ is colored yellow. Other conserved residues are indicated by an asterisk. Note the minimal sequence variation between Hfq of E. coli serotypes (lines 1–5), Vibrio vulnificus (line 6), Vibrio harveyi (line 7), Vibrio parahaemolyticus (line 8), Vibrio fischeri (line 9), N. gonorrhoeae strains (lines 10 and 11), N. meningitidis strains (lines 12-15) and N. lactamica (line 16) (adapted from Schumacher et al., 2002).
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fig01: Amino acid sequence alignment of Hfq proteins of Escherichia coli serotypes, Vibrio species, Neisseria meningitidis, Neisseria gonorrhoeae strains and Neisseria lactamica. The secondary structural elements of Hfq protein are shown above the alignment with the N-terminal helix α1 in cyan. The Sm1 and Sm2 motif regions are boxed. The sole conserved residue Gly34 is indicated in red. Highly conserved hydrophobic residues within the Sm1 region are indicated by a lower case h, and the two highly conserved glycine and aspartic acid residues within the Sm1 motif are indicated by G and D, respectively. The absolutely conserved glutamine of helix α1 that is important for base recognition and the highly conserved tyrosine (or phenylalanine) are indicated in green. Within the Sm2 region, the ‘Hfq Sm2 motif YKH’ is colored yellow. Other conserved residues are indicated by an asterisk. Note the minimal sequence variation between Hfq of E. coli serotypes (lines 1–5), Vibrio vulnificus (line 6), Vibrio harveyi (line 7), Vibrio parahaemolyticus (line 8), Vibrio fischeri (line 9), N. gonorrhoeae strains (lines 10 and 11), N. meningitidis strains (lines 12-15) and N. lactamica (line 16) (adapted from Schumacher et al., 2002).

Mentions: The complete genome sequences of four meningococcal, two strains of the closely related human pathogen Neisseria gonorrhoeae and one commensal neisserial species (Neisseria lactamica) are known (Parkhill et al., 2000; Tettelin et al., 2000; Bentley et al., 2007; Chung et al., 2008; Peng et al., 2008). The amino acid sequence of Hfq of the meningococcal strain used in this study (H44/76) is identical to the Hfq sequence of meningococcal strain FAM18 and 98% identical to the sequences of the other three meningococcal strains. In addition, the sequence of H44/76 is 98% identical to gonococcal Hfq and 95% identical to the Hfq sequence of N. lactamica. The H44/76 neisserial Hfq amino acid sequence shows 63% and 60% identities to Hfq proteins of E. coli and Vibrio spp., respectively (Fig. 1). Of importance, hardly any amino acid substitutions in the meningococcal Hfq sequence, compared with those of E. coli and Vibro spp., are observed in highly conserved regions of the protein shown to be important for functionality (Fig. 1) (Schumacher et al., 2002).


Molecular characterization and identification of proteins regulated by Hfq in Neisseria meningitidis.

Pannekoek Y, Huis in 't Veld R, Hopman CT, Langerak AA, Speijer D, van der Ende A - FEMS Microbiol. Lett. (2009)

Amino acid sequence alignment of Hfq proteins of Escherichia coli serotypes, Vibrio species, Neisseria meningitidis, Neisseria gonorrhoeae strains and Neisseria lactamica. The secondary structural elements of Hfq protein are shown above the alignment with the N-terminal helix α1 in cyan. The Sm1 and Sm2 motif regions are boxed. The sole conserved residue Gly34 is indicated in red. Highly conserved hydrophobic residues within the Sm1 region are indicated by a lower case h, and the two highly conserved glycine and aspartic acid residues within the Sm1 motif are indicated by G and D, respectively. The absolutely conserved glutamine of helix α1 that is important for base recognition and the highly conserved tyrosine (or phenylalanine) are indicated in green. Within the Sm2 region, the ‘Hfq Sm2 motif YKH’ is colored yellow. Other conserved residues are indicated by an asterisk. Note the minimal sequence variation between Hfq of E. coli serotypes (lines 1–5), Vibrio vulnificus (line 6), Vibrio harveyi (line 7), Vibrio parahaemolyticus (line 8), Vibrio fischeri (line 9), N. gonorrhoeae strains (lines 10 and 11), N. meningitidis strains (lines 12-15) and N. lactamica (line 16) (adapted from Schumacher et al., 2002).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2734931&req=5

fig01: Amino acid sequence alignment of Hfq proteins of Escherichia coli serotypes, Vibrio species, Neisseria meningitidis, Neisseria gonorrhoeae strains and Neisseria lactamica. The secondary structural elements of Hfq protein are shown above the alignment with the N-terminal helix α1 in cyan. The Sm1 and Sm2 motif regions are boxed. The sole conserved residue Gly34 is indicated in red. Highly conserved hydrophobic residues within the Sm1 region are indicated by a lower case h, and the two highly conserved glycine and aspartic acid residues within the Sm1 motif are indicated by G and D, respectively. The absolutely conserved glutamine of helix α1 that is important for base recognition and the highly conserved tyrosine (or phenylalanine) are indicated in green. Within the Sm2 region, the ‘Hfq Sm2 motif YKH’ is colored yellow. Other conserved residues are indicated by an asterisk. Note the minimal sequence variation between Hfq of E. coli serotypes (lines 1–5), Vibrio vulnificus (line 6), Vibrio harveyi (line 7), Vibrio parahaemolyticus (line 8), Vibrio fischeri (line 9), N. gonorrhoeae strains (lines 10 and 11), N. meningitidis strains (lines 12-15) and N. lactamica (line 16) (adapted from Schumacher et al., 2002).
Mentions: The complete genome sequences of four meningococcal, two strains of the closely related human pathogen Neisseria gonorrhoeae and one commensal neisserial species (Neisseria lactamica) are known (Parkhill et al., 2000; Tettelin et al., 2000; Bentley et al., 2007; Chung et al., 2008; Peng et al., 2008). The amino acid sequence of Hfq of the meningococcal strain used in this study (H44/76) is identical to the Hfq sequence of meningococcal strain FAM18 and 98% identical to the sequences of the other three meningococcal strains. In addition, the sequence of H44/76 is 98% identical to gonococcal Hfq and 95% identical to the Hfq sequence of N. lactamica. The H44/76 neisserial Hfq amino acid sequence shows 63% and 60% identities to Hfq proteins of E. coli and Vibrio spp., respectively (Fig. 1). Of importance, hardly any amino acid substitutions in the meningococcal Hfq sequence, compared with those of E. coli and Vibro spp., are observed in highly conserved regions of the protein shown to be important for functionality (Fig. 1) (Schumacher et al., 2002).

Bottom Line: Using proteomics, the expression of 28 proteins was found to be significantly affected upon deletion of hfq.One of the differentially expressed proteins, GdhA, was identified as an essential virulence factor for establishment of sepsis in an animal model, studied earlier.These results show that in N. meningitidis Hfq is involved in the regulation of a variety of components contributing to the survival and establishment of meningococcal disease.

View Article: PubMed Central - PubMed

ABSTRACT
Hfq is a highly conserved pleiotropically acting prokaryotic RNA-binding protein involved in the post-transcriptional regulation of many stress-responsive genes by small RNAs. In this study, we show that Hfq of the strictly human pathogen Neisseria meningitidis is involved in the regulation of expression of components involved in general metabolic pathways, iron metabolism and virulence. A meningococcal hfq deletion strain (H44/76Deltahfq) is impaired in growth in nutrient-rich media and does not grow at all in nutrient-limiting medium. The growth defect was complemented by expression of hfq in trans. Using proteomics, the expression of 28 proteins was found to be significantly affected upon deletion of hfq. Of these, 20 proteins are involved in general metabolism, among them seven iron-responsive genes. Two proteins (PilE, TspA) are involved in adherence to human cells, a step crucial for the onset of disease. One of the differentially expressed proteins, GdhA, was identified as an essential virulence factor for establishment of sepsis in an animal model, studied earlier. These results show that in N. meningitidis Hfq is involved in the regulation of a variety of components contributing to the survival and establishment of meningococcal disease.

Show MeSH
Related in: MedlinePlus