Recovery of <i>A. baumannii sod2343</i> mutant from <i>Galleria mellonella</i> larvae is minor compared to parental strain.

<p>Groups of 10 larvae were injected with 1.5×10⁶ CFU of ATCC 17978, ATCC 17978 <i>sod2343::Km</i>, and ATCC 17978 <i>sod2343::Km</i> pWH<i>sod2343</i>, respectively. Directly after injection of the 10 larvae within a group, the larvae were frozen at −80°C, homogenized and serial dilutions were plated on CHROMagar Acinetobacter to determine CFUs. Boxes indicate the 25–75% region, the median is indicated by a line in the box, the average is indicated by a small square, whiskers indicate maximum and minimum values, respectively.</p

Minimal inhibitory concentrations determined by Etest.

<p>Average MIC values in [µg/ml] determined from three (MIC³) and six (MIC⁶) independent experiments, respectively; MIC value given in <b>bold</b> indicates that MIC value of mutant is significantly different from MIC value of the corresponding parental strain (p<0.05; Student's t-test, two-tailed, unpaired;</p><p>* indicates p<0.005).</p

<i>A. baumannii sod2343</i> mutants are attenuated in the <i>Galleria mellonella</i> infection model.

<p>(A) Survival of <i>Galleria</i> caterpillars injected with 3×10⁵ CFU of ATCC 17978 <i>sod2343::Km</i> (red), ATCC 17978 (green) and PBS (blue). (B) Infection with 1.5×10⁶ CFU of 07–095 and its <i>sod2343::Km</i> derivative. (C) Infection with 3×10⁵ CFU of 07–102 and its <i>sod2343::Km</i> mutant. Results represent means and standard deviations of at least three independent experiments of 16 larvae per group.</p

Confirmation of <i>sod2343::Km</i> mutants by immunoblotting.

<p>Overnight cultures as indicated were diluted 1∶20, grown for another 4 hours at 37°C, then adjusted to 1 OD (600 nm) and 0.5 ml of each was centrifuged and the pellet resuspended in 50 µl of loading buffer. 10 µl of each sample was loaded on an SDS-PAGE that was subsequently electro-blotted. A polyclonal antiserum raised against GST-SOD2343 fusion protein was diluted 1∶5000 for detection. The blots shown are representative of three independent replicates.</p

Growth defects of <i>sod2343</i> mutants depend on extent of aeration.

<p>Bacterial cultures as indicated were OD-adjusted from overnight cultures and grown at 37°C in LB medium under constant shaking for 8 hours either in baffled flasks (A) or in tubes (B) and the OD (600 nm) was determined. For each strain, data obtained from three independent cultures were averaged; error bars represent plus/minus one standard deviation. Growth of all <i>sod2343</i> mutants was significantly delayed compared to their parentals when grown in baffled flasks (A) (p<0.005, Student's <i>t</i> test,two-tailed, unpaired, for the last three time points). When grown in tubes (B), only the difference between ATCC 17978 and ATCC 17978 <i>sod2343</i> was significant (p<0.05).</p

DataSheet_6_Protein domain-dependent vesiculation of Lipoprotein A, a protein that is important in cell wall synthesis and fitness of the human respiratory pathogen Haemophilus influenzae.docx

The human pathogen Haemophilus influenzae causes respiratory tract infections and is commonly associated with prolonged carriage in patients with chronic obstructive pulmonary disease. Production of outer membrane vesicles (OMVs) is a ubiquitous phenomenon observed in Gram-negative bacteria including H. influenzae. OMVs play an important role in various interactions with the human host; from neutralization of antibodies and complement activation to spread of antimicrobial resistance. Upon vesiculation certain proteins are found in OMVs and some proteins are retained at the cell membrane. The mechanism for this phenomenon is not fully elucidated. We employed mass spectrometry to study vesiculation and the fate of proteins in the outer membrane. Functional groups of proteins were differentially distributed on the cell surface and in OMVs. Despite its supposedly periplasmic and outer membrane location, we found that the peptidoglycan synthase-activator Lipoprotein A (LpoA) was accumulated in OMVs relative to membrane fractions. A mutant devoid of LpoA lost its fitness as revealed by growth and electron microscopy. Furthermore, high-pressure liquid chromatography disclosed a lower concentration (55%) of peptidoglycan in the LpoA-deficient H. influenzae compared to the parent wild type bacterium. Using an LpoA-mNeonGreen fusion protein and fluorescence microscopy, we observed that LpoA was enriched in “foci” in the cell envelope, and further located in the septum during cell division. To define the fate of LpoA, C-terminally truncated LpoA-variants were constructed, and we found that the LpoA C-terminal domain promoted optimal transportation to the OMVs as revealed by flow cytometry. Taken together, our study highlights the importance of LpoA for H. influenzae peptidoglycan biogenesis and provides novel insights into cell wall integrity and OMV production.</p

DataSheet_1_Protein domain-dependent vesiculation of Lipoprotein A, a protein that is important in cell wall synthesis and fitness of the human respiratory pathogen Haemophilus influenzae.docx

The human pathogen Haemophilus influenzae causes respiratory tract infections and is commonly associated with prolonged carriage in patients with chronic obstructive pulmonary disease. Production of outer membrane vesicles (OMVs) is a ubiquitous phenomenon observed in Gram-negative bacteria including H. influenzae. OMVs play an important role in various interactions with the human host; from neutralization of antibodies and complement activation to spread of antimicrobial resistance. Upon vesiculation certain proteins are found in OMVs and some proteins are retained at the cell membrane. The mechanism for this phenomenon is not fully elucidated. We employed mass spectrometry to study vesiculation and the fate of proteins in the outer membrane. Functional groups of proteins were differentially distributed on the cell surface and in OMVs. Despite its supposedly periplasmic and outer membrane location, we found that the peptidoglycan synthase-activator Lipoprotein A (LpoA) was accumulated in OMVs relative to membrane fractions. A mutant devoid of LpoA lost its fitness as revealed by growth and electron microscopy. Furthermore, high-pressure liquid chromatography disclosed a lower concentration (55%) of peptidoglycan in the LpoA-deficient H. influenzae compared to the parent wild type bacterium. Using an LpoA-mNeonGreen fusion protein and fluorescence microscopy, we observed that LpoA was enriched in “foci” in the cell envelope, and further located in the septum during cell division. To define the fate of LpoA, C-terminally truncated LpoA-variants were constructed, and we found that the LpoA C-terminal domain promoted optimal transportation to the OMVs as revealed by flow cytometry. Taken together, our study highlights the importance of LpoA for H. influenzae peptidoglycan biogenesis and provides novel insights into cell wall integrity and OMV production.</p

DataSheet_2_Protein domain-dependent vesiculation of Lipoprotein A, a protein that is important in cell wall synthesis and fitness of the human respiratory pathogen Haemophilus influenzae.docx

The human pathogen Haemophilus influenzae causes respiratory tract infections and is commonly associated with prolonged carriage in patients with chronic obstructive pulmonary disease. Production of outer membrane vesicles (OMVs) is a ubiquitous phenomenon observed in Gram-negative bacteria including H. influenzae. OMVs play an important role in various interactions with the human host; from neutralization of antibodies and complement activation to spread of antimicrobial resistance. Upon vesiculation certain proteins are found in OMVs and some proteins are retained at the cell membrane. The mechanism for this phenomenon is not fully elucidated. We employed mass spectrometry to study vesiculation and the fate of proteins in the outer membrane. Functional groups of proteins were differentially distributed on the cell surface and in OMVs. Despite its supposedly periplasmic and outer membrane location, we found that the peptidoglycan synthase-activator Lipoprotein A (LpoA) was accumulated in OMVs relative to membrane fractions. A mutant devoid of LpoA lost its fitness as revealed by growth and electron microscopy. Furthermore, high-pressure liquid chromatography disclosed a lower concentration (55%) of peptidoglycan in the LpoA-deficient H. influenzae compared to the parent wild type bacterium. Using an LpoA-mNeonGreen fusion protein and fluorescence microscopy, we observed that LpoA was enriched in “foci” in the cell envelope, and further located in the septum during cell division. To define the fate of LpoA, C-terminally truncated LpoA-variants were constructed, and we found that the LpoA C-terminal domain promoted optimal transportation to the OMVs as revealed by flow cytometry. Taken together, our study highlights the importance of LpoA for H. influenzae peptidoglycan biogenesis and provides novel insights into cell wall integrity and OMV production.</p

Table_2_Protein domain-dependent vesiculation of Lipoprotein A, a protein that is important in cell wall synthesis and fitness of the human respiratory pathogen Haemophilus influenzae.docx

The human pathogen Haemophilus influenzae causes respiratory tract infections and is commonly associated with prolonged carriage in patients with chronic obstructive pulmonary disease. Production of outer membrane vesicles (OMVs) is a ubiquitous phenomenon observed in Gram-negative bacteria including H. influenzae. OMVs play an important role in various interactions with the human host; from neutralization of antibodies and complement activation to spread of antimicrobial resistance. Upon vesiculation certain proteins are found in OMVs and some proteins are retained at the cell membrane. The mechanism for this phenomenon is not fully elucidated. We employed mass spectrometry to study vesiculation and the fate of proteins in the outer membrane. Functional groups of proteins were differentially distributed on the cell surface and in OMVs. Despite its supposedly periplasmic and outer membrane location, we found that the peptidoglycan synthase-activator Lipoprotein A (LpoA) was accumulated in OMVs relative to membrane fractions. A mutant devoid of LpoA lost its fitness as revealed by growth and electron microscopy. Furthermore, high-pressure liquid chromatography disclosed a lower concentration (55%) of peptidoglycan in the LpoA-deficient H. influenzae compared to the parent wild type bacterium. Using an LpoA-mNeonGreen fusion protein and fluorescence microscopy, we observed that LpoA was enriched in “foci” in the cell envelope, and further located in the septum during cell division. To define the fate of LpoA, C-terminally truncated LpoA-variants were constructed, and we found that the LpoA C-terminal domain promoted optimal transportation to the OMVs as revealed by flow cytometry. Taken together, our study highlights the importance of LpoA for H. influenzae peptidoglycan biogenesis and provides novel insights into cell wall integrity and OMV production.</p

DataSheet_4_Protein domain-dependent vesiculation of Lipoprotein A, a protein that is important in cell wall synthesis and fitness of the human respiratory pathogen Haemophilus influenzae.docx

The human pathogen Haemophilus influenzae causes respiratory tract infections and is commonly associated with prolonged carriage in patients with chronic obstructive pulmonary disease. Production of outer membrane vesicles (OMVs) is a ubiquitous phenomenon observed in Gram-negative bacteria including H. influenzae. OMVs play an important role in various interactions with the human host; from neutralization of antibodies and complement activation to spread of antimicrobial resistance. Upon vesiculation certain proteins are found in OMVs and some proteins are retained at the cell membrane. The mechanism for this phenomenon is not fully elucidated. We employed mass spectrometry to study vesiculation and the fate of proteins in the outer membrane. Functional groups of proteins were differentially distributed on the cell surface and in OMVs. Despite its supposedly periplasmic and outer membrane location, we found that the peptidoglycan synthase-activator Lipoprotein A (LpoA) was accumulated in OMVs relative to membrane fractions. A mutant devoid of LpoA lost its fitness as revealed by growth and electron microscopy. Furthermore, high-pressure liquid chromatography disclosed a lower concentration (55%) of peptidoglycan in the LpoA-deficient H. influenzae compared to the parent wild type bacterium. Using an LpoA-mNeonGreen fusion protein and fluorescence microscopy, we observed that LpoA was enriched in “foci” in the cell envelope, and further located in the septum during cell division. To define the fate of LpoA, C-terminally truncated LpoA-variants were constructed, and we found that the LpoA C-terminal domain promoted optimal transportation to the OMVs as revealed by flow cytometry. Taken together, our study highlights the importance of LpoA for H. influenzae peptidoglycan biogenesis and provides novel insights into cell wall integrity and OMV production.</p