The lytic transglycosylase MltB connects membrane homeostasis and in vivo fitness of Acinetobacter baumannii

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/146339/1/mmi14000-sup-0001-Supinfo.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/146339/2/mmi14000.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/146339/3/mmi14000_am.pd

Proteobactin and a yersiniabactin-related siderophore mediate iron acquisition in Proteus mirabilis

Proteus mirabilis causes complicated urinary tract infections (UTIs). While the urinary tract is an iron-limiting environment, iron acquisition remains poorly characterized for this uropathogen. Microarray analysis of P. mirabilis HI4320 cultured under iron limitation identified 45 significantly upregulated genes ( P  ≤  0.05) that represent 21 putative iron-regulated systems. Two gene clusters, PMI0229-0239 and PMI2596-2605, encode putative siderophore systems. PMI0229-0239 encodes a non-ribosomal peptide synthetase-independent siderophore system for producing a novel siderophore, proteobactin. PMI2596-2605 are contained within the high-pathogenicity island, originally described in Yersinia pestis , and encodes proteins with apparent homology and organization to those involved in yersiniabactin production and uptake. Cross-feeding and biochemical analysis shows that P. mirabilis is unable to utilize or produce yersiniabactin, suggesting that this yersiniabactin-related locus is functionally distinct. Only disruption of both systems resulted in an in vitro iron-chelating defect; demonstrating production and iron-chelating activity for both siderophores. These findings clearly show that proteobactin and the yersiniabactin-related siderophore function as iron acquisition systems. Despite the activity of both siderophores, only mutants lacking the yersiniabactin-related siderophore have reduced fitness in vivo . The fitness requirement for the yersiniabactin-related siderophore during UTI shows, for the first time, the importance of siderophore production in vivo for P. mirabilis .Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/79111/1/MMI_7317_sm_FigS1-S2_TabS1-S3.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/79111/2/j.1365-2958.2010.07317.x.pd

Contribution of the TCA cycle and gluconeogenesis during UTI.

<p>Competitive indices (CI) were determined following co-challenge infections of female CBA/J mice with a 1∶1 ratio of either wild-type (A) <i>E. coli</i> CFT073 or (B) <i>P. mirabilis</i> HI4320 and their respective mutants in the following genes: <i>sdhB</i>, succinate dehydrogenase; <i>fumC</i>, fumarate hydratase; <i>frdA</i>, fumarate reductase; and <i>pckA</i>, phosphoenolpyruvate carboxykinase. <i>E. coli</i> was cultured from bladders and kidneys at 48 hpi. <i>P. mirabilis</i> was cultured from organs at 7 dpi. Each dot represents bladder (closed symbols) and kidneys (open symbols) from an individual animal. Bars indicate the median CI. Significant differences in colonization (*P<0.05) were determined by the Wilcoxon signed-rank test. A CI<1 indicates a fitness defect. Growth of (C, E) <i>E. coli</i> CFT073 and (D, F) <i>P. mirabilis</i> HI4320 wild-type and mutant strains in: <i>sdhB</i>, <i>fumC</i>, <i>frdA</i>, and <i>pckA</i> in LB medium (C, D) or defined medium containing 0.2% glucose (E, F) as the carbon source. A representative growth curve is shown for each panel.</p

Model describing the differential effect of <i>E. coli</i> and <i>P. mirabilis</i> metabolism on the C/N ratio within the urinary tract.

<p>The urinary tract environment has a low C/N ratio due to the dilute mixture of amino acids and peptides as the primary carbon source and the abundance of urea in urine providing a substantial nitrogen contribution. <i>E. coli</i> is unable to utilize or sense the nitrogen sequestered in urea because it lacks urease, which liberates ammonia from urea. In contrast, <i>P. mirabilis</i> is urease positive; consequently, <i>P. mirabilis</i> senses a physiologically lower C/N ratio than <i>E. coli</i>. This results in <i>E. coli</i> activation of the glutamine synthetase and glutamate oxo-glutarate aminotransferase system (GS/GOGAT) to assimilate nitrogen while <i>P. mirabilis</i> assimilates nitrogen, via glutamate dehydrogenase (Gdh) due to the apparent excess nitrogen available from ammonia produced by urea hydrolysis. This difference in physiological nitrogen availability explains the dramatic difference between <i>E. coli</i> and <i>P. mirabilis</i> central carbon pathway requirements for fitness during urinary tract infection.</p

Diagram of central metabolism and map of the specific pathways disrupted by targeted mutations in uropathogenic <i>E. coli</i> and <i>P. mirabilis</i>.

<p>Carbon sources or biochemical intermediates shared between pathways are indicated in capital letters or abbreviated: G6P, glucose-6-phosphate; F6P, fructose-6-phosphate; G3P, glyceraldehyde-3-phosphate; 6PGN, 6-phosphogluconate. Reactions are denoted with arrows. Specific reactions (red arrows) were targeted by deletion or insertion in <i>E. coli</i> CFT073 or <i>P. mirabilis</i> HI4320, respectively. In glycolysis: <i>pgi</i>, glucose-6-phosphate isomerase; <i>pfkA</i>, 6-phosphofructokinase transferase; <i>tpiA</i>, triosephosphate isomerase; <i>pykA</i>, pyruvate kinase; in pentose phosphate pathway: gnd, 6-phosphogluconate dehydrogenase; <i>talB</i>, transaldolase; in Entner-Duodoroff pathway: <i>edd</i>, 6-phosphogluconate dehydratase; in gluconeogenesis: <i>pckA</i>, phosphoenolpyruvate carboxykinase; and in the TCA cycle: <i>sdhB</i>, succinate dehydrogenase; <i>fumC</i>, fumarate hydratase; <i>frdA</i>, fumarate reductase.</p

<i>In vivo</i> role for the pentose phosphate pathway and the Entner-Doudoroff pathway for pathogen colonization of the urinary tract.

<p>Competitive indices (CI) were determined following co-challenge infections of female CBA/J mice with a 1∶1 ratio of either wild-type (A) UPEC CFT073 or (B) <i>P. mirabilis</i> HI4320 and their respective mutants in the following genes: gnd, 6-phosphogluconate dehydrogenase; <i>talB</i>, transaldolase; and <i>edd</i>, 6-phosphoglyconate dehydrase. The contribution of multiple transaldolase isoenzymes in <i>E. coli</i> were assessed using co-challenge infections with wild-type <i>E. coli</i> CFT073 and (C) <i>talA</i> or (D) <i>talAtalB</i> mutant strains. <i>E. coli</i> CFT073 was cultured from bladders and kidneys at 48 hpi. <i>P. mirabilis</i> HI4320 was cultured from organs at 7 dpi. Each dot represents bladder (closed symbols) and kidneys (open symbols) from an individual animal. Bars indicate the median CI. Significant differences in colonization (*P<0.05) were determined by Wilcoxon signed-rank test. A CI<1 indicates a fitness defect.</p

Glycolysis is required for <i>P. mirabilis</i> but is dispensible for <i>E. coli</i> during experimental urinary tract infection.

<p><i>In vivo</i> competitive indices (CI) were determined following co-challenge infections of susceptible female CBA/J mice with a 1∶1 ratio of either wild-type (A) <i>E. coli</i> CFT073 or (B) <i>P. mirabilis</i> HI4320 and their respective glycolysis mutant strains. <i>E. coli</i> were recovered at 48 h post-inoculation (hpi). <i>P. mirabilis</i> was recovered at 7 d post-inoculation (dpi). Each dot represents bladder (closed symbols) and kidneys (open symbols) from an individual animal. Bars indicate the median CI. Significant differences in colonization (*P<0.05) were determined with the Wilcoxon signed-rank test. A CI<1 indicates a fitness defect. Growth of (C, E) <i>E. coli</i> CFT073 or (D, F) <i>P. mirabilis</i> HI4320 wild-type strains and mutants in: <i>pgi</i>, glucose-6-phosphate isomerase; <i>pfkA</i>, 6-phosphofructokinase transferase; <i>tpiA</i>, triosephosphate isomerase; and <i>pykA</i>, pyruvate kinase during culture in defined medium containing 0.2% glucose as the sole carbon source (C, D) or in LB medium (E, F). A representative growth curve is shown for each panel. Restoration of wild-type growth in triosephosphate isomerase mutants (G) growth in MOPS defined medium containing 0.2% glucose of <i>E. coli</i> CFT073 (pGEN), UPEC <i>tpiA</i> (pGEN), and the <i>E. coli</i> CFT073 <i>tpiA</i> mutant containing pGEN into which the <i>tpiA</i> gene from <i>P. mirabilis</i> was cloned. (H) <i>P. mirabilis</i> HI4320 containing empty vector (pGEN), HI4320 <i>tpiA</i> (pGEN), and complemented HI4320 <i>tpiA</i> mutant containing pGEN-<i>tpiA</i> in minimal salts medium containing 0.2% glucose. (I) <i>In vivo</i> complementation of <i>P. mirabilis tpiA</i> mutant bacteria. Competitive indices were determined following an <i>in vivo</i> co-challenge infection of female CBA/J mice with <i>P. mirabilis</i> HI4320 (pGEN) and HI4320 (<i>tpiA</i> pGEN-<i>tpiA</i>). Bacteria were recovered at 7 dpi. Each dot represents the CI in bladders (closed symbols) and kidneys (open symbols) from an individual animal. Bars represent the median CI. P-values are indicated on the graph.</p

Polymicrobial urinary tract infection with <i>E. coli</i> and <i>P. mirabilis</i> enhances persistent bacterial colonization.

<p>Colonization levels following independent infections of female CBA/J mice with (A) UPEC strain CFT073 or (B) <i>P. mirabilis</i> HI4320. Colonization levels at (C) 48 hpi and (D) 7 dpi following polymicrobial infection of female CBA/J mice inoculated with a mixture of CFT073 and HI4320. The CFU/g of tissue for UPEC (circles) and <i>P. mirabilis</i> (squares) from bladders (closed symbols) and kidneys (open symbols) from individual animals at 48 hpi for (C) or 7 dpi for (A, B, D). Bars indicate the median values.</p

Polymicrobial infection alters central metabolism requirements for <i>E. coli</i> and <i>P. mirabilis</i>.

<p>(A) Competitive indices (CI) were determined following co-challenge infections of female CBA/J mice with a 1∶1 ratio of wild-type: mutant bacteria for <i>gnd</i> (oxidative pentose phosphate pathway) and <i>pckA</i> (gluconeogenesis) for <i>P. mirabilis</i> at 48 hpi. Each dot represents bladder (closed symbols) and kidneys (open symbols) from an individual animal. Competitive indices (CI) were determined 48 hpi for mixed infections of (B) wild-type <i>E. coli</i> CFT073 and <i>P. mirabilis</i> HI4320 <i>gnd</i>, (C) wild-type HI4320 and CFT073 <i>gnd</i>, and (D) HI4320 <i>gnd</i> and CFT073 <i>gnd</i> mutant constructs. Each circle represents bladder or kidneys from an individual animal. In (A–D) bars indicate the median CI and significant differences in colonization (*) (P<0.05) were determined by Wilcoxon signed-rank test. (E) <i>In vivo</i> CI at 48 h and 7 d post-infection. (F) CI during logarithmic growth in LB medium. For (A–F) a CI<1 indicates a fitness defect. For mixed infections CFU/ml were determined following plating of serial dilutions on LB agar with and without tetracycline. CFU from tetracycline-containing plates (<i>P. mirabilis</i> are Tet^R) were subtracted from total CFU recovered on LB agar without antibiotics to determine CFU/ml for <i>E. coli</i> (Tet^S).</p