The glycosyl hydrolase activity of <i>S. pneumoniae</i> GHIP.

<p>(A) <i>S. pneumoniae</i> GHIP (green) superimposed on its homolog from <i>Bacillus anthracis</i>, PlyB (cyan) (PDB code 2NW0). The magenta arrow points toward the major difference, where the N-terminal regions form a helix (α1) which is absent in PlyB. The sticks represent the four key acidic residues to their enzyme activities, inculding Asp56, Asp154, Glu156, and Asp245 of GHIP and Asp6, Asp90, Asp92, and Asp171 of PlyB. (B) The enlarged image of the four key residues at the active site. Close up view of <i>S. pneumoniae</i> GHIP showing overlay of key acidic residues: Asp56, Asp154, Glu156, and Asp245 of GHIP and Asp6, Asp90, Asp92, and Asp171 of PlyB, which exhibit similar locations and orientations. (C) The month of TIM barrel in <i>S. pneumoniae</i> GHIP is active site which contains 14 residues in sticks, including Asp56, Ser58, Ser84, Tyr121, Tyr123, Glu154, Glu156, Asp157, Tyr185, Tyr209, Asp212, Ser233, Asp243 and Asp245. The GHIP structure is shown in the context of a transparent (gray) surface. (D) The enlarged image of the 14 residues at the active site and the background is the electrostatic potential surface of <i>S. pneumoniae</i> GHIP. Saturated red indicates Φ<−10 kiloteslas/e, and saturated blue indicates Φ>10 kiloteslas/e, T = 20°C. (E) & (F) The temperature and pH activity analyses of <i>S. pneumoniae</i> GHIP. Peptidoglycan hydrolase activity was measured at various pH (4.0 to 8.0) and temperatures (25 to 45°C) ranges as described in the Materials and Methods. (G) Hydrolase activity of <i>S. pneumoniae</i> GHIP on PNP-(GlcNAc)₅. Lane 1 represents the positive control using HEWL (egg white lysozyme); lane 2 is native GHIP; lanes 3–6 are active-site mutants D56A, D154A, E156A, and D245A, respectively. Asterisks denote values significantly different from the wild-type strain by Student’s <i>t</i>-test (*, P<0.05; **, P<0.01).</p

X-ray data collection and refinement statistics of <i>S. pneumoniae</i> GHIP.

a<p>Values in parentheses are for the highest-resolution shells.</p>b<p>R_merge = ∑(I−<i>)/∑I, where I is the observed intensity and <i> is the statistically weighted average intensity of multiple symmetry related observation.</i></i></p><i><i>c<p>R_factor: R = ∑||F_calc|−|F_obs|/∑|F_obs|, where F_calc and F_obs are the calculated and observed structure factors, respectively. R_free is calculated the same as R_factor but from a test set containing 5% of data excluded from the refinement calculation.</p>d<p>rmsd, root-mean-square deviation from ideal geometry.</p></i></i

The overall structure of <i>S. pneumoniae</i> GHIP.

<p>(A) The structure of <i>S. pneumoniae</i> GHIP in stereo view. (B) The topological diagram of <i>S. pneumoniae</i> GHIP. The barrels and arrows indicate the helix and β-strand structures. (C) The electrostatic potential surface of <i>S. pneumoniae</i> GHIP. Saturated red indicates Φ<−10 kiloteslas/e, and saturated blue indicates Φ>10 kiloteslas/e, T = 20°C. The magenta arrow points towards the negatively charged groove.</p

Adherence and invasion of the <i>ΔGHIP</i> mutant into host cells <i>in vitro</i>.

<p>(A) & (B) <i>S. pneumoniae</i> type R6 and their isogenic R6Δ<i>GHIP</i> mutants were examined for adherence to A549 and CNE2 cells, respectively. (C) & (D) <i>S. pneumoniae</i> type R6 and their isogenic R6Δ<i>GHIP</i> were examined for invasion into A549 and CNE2 cells, respectively. Infection experiments were conducted for 4 h at 37°C. Wild-type <i>S. pneumoniae</i> and GHIP-deficient mutants were generated to be devoid of pneumolysin in order to avoid destruction of the monolayer in the <i>in vitro</i> infection system. Scoring the number of adherent and invasive bacteria indicate that adhesion and invasion are substantially reduced for GHIP-deficient <i>S. pneumoniae</i>. Results are presented as the mean ± standard deviation for at least three independent experiments. Asterisks and triangles denote values significantly different from wild-type by Student’s <i>t</i>-test (**, P<0.01; ▴, P<0.05).</p

Animal and colonization experiments testing <i>S. pneumoniae</i> GHIP function.

<p>(A) & (B) The effect of the <i>GHIP</i> deletion mutation on virulence. Groups of 18 BALB/c mice were intranasally or intraperitoneally challenged with 1.0 × 10⁸ and 1.0 × 10³ CFU, respectively, of D39 or the isogenic <i>ΔGHIP</i> mutant. Each datum point represents one mouse. Solid line, wild-type D39; dotted line, <i>ΔGHIP</i> mutant. Asterisks denote values significantly different from wild-type by Student’s <i>t-</i>test (*, P<0.05). (C) The effect of the <i>GHIP</i> deletion mutation on bacteria recovered from nasopharynxes, lungs, and blood samples of BALB/c mice after intranasal challenge. BALB/c mice were intranasally challenged with either wild-type D39 or the <i>ΔGHIP</i> mutant at 1.0 × 10⁸CFU/mouse. At 12, 24, and 36 h post-infection, six mice from each group were scarified, and the number of recovered bacteria was determined by plating on blood agar. Results are expressed as log₁₀ CFUs per gram tissue and represent individually recorded values for each mouse. Horizontal bars represent geometric means. Asterisks denote values significantly different from wild-type by Student’s <i>t-</i>test (*, P<0.05).</p

Amino-acid sequence alignment comparing homologs of <i>S.pneumoniae</i> GHIP.

<p>Sequence alignment of five streptococcus SpGHIP homologs, including SpGHIP from <i>Streptococcus pneumoniae</i> TIGR4 (accession number NP_345466), <i>Streptococcus gordonii</i> (accession number YP_001450056), <i>Streptococcus sanguinis</i> (accession number EGD37983), <i>Streptococcus pyogenes</i> (accession number NP_268858), <i>Streptococcus suis</i> (accession number EHC03477), and PlyB from the BcpI phage (PDB code 2NW0). Identical and similar residues are shown in <i>cyan</i> and <i>yellow</i>, respectively. Secondary structure elements of SpGHIP are shown above the sequences. Residues are numbered according to the SpGHIP sequence. Sequence alignment was generated using ClustalW.</p