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Burkholderia pseudomallei known siderophores and hemin uptake are dispensable for lethal murine melioidosis.

Kvitko BH, Goodyear A, Propst KL, Dow SW, Schweizer HP - PLoS Negl Trop Dis (2012)

Bottom Line: Prolonged incubation of a hmu hem mutant in hemoglobin-containing minimal medium yielded variants able to utilize hemoglobin and hemin suggesting alternate pathways for utilization of these two host iron sources.Lactoferrin utilization was dependent on malleobactin, but not pyochelin synthesis and/or uptake.These data suggest that B. pseudomallei may employ a novel ferritin-iron acquisition pathway as a means to sustain in vivo growth.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Immunology and Pathology, Rocky Mountain Regional Center of Excellence for Biodefense and Emerging Infectious Diseases Research, Colorado State University, Fort Collins, Colorado, United States of America.

ABSTRACT
Burkholderia pseudomallei is a mostly saprophytic bacterium, but can infect humans where it causes the difficult-to-manage disease melioidosis. Even with proper diagnosis and prompt therapeutic interventions mortality rates still range from >20% in Northern Australia to over 40% in Thailand. Surprisingly little is yet known about how B. pseudomallei infects, invades and survives within its hosts, and virtually nothing is known about the contribution of critical nutrients such as iron to the bacterium's pathogenesis. It was previously assumed that B. pseudomallei used iron-acquisition systems commonly found in other bacteria, for example siderophores. However, our previous discovery of a clinical isolate carrying a large chromosomal deletion missing the entire malleobactin gene cluster encoding the bacterium's major high-affinity siderophore while still being fully virulent in a murine melioidosis model suggested that other iron-acquisition systems might make contributions to virulence. Here, we deleted the major siderophore malleobactin (mba) and pyochelin (pch) gene clusters in strain 1710b and revealed a residual siderophore activity which was unrelated to other known Burkholderia siderophores such as cepabactin and cepaciachelin, and not due to increased secretion of chelators such as citrate. Deletion of the two hemin uptake loci, hmu and hem, showed that Hmu is required for utilization of hemin and hemoglobin and that Hem cannot complement a Hmu deficiency. Prolonged incubation of a hmu hem mutant in hemoglobin-containing minimal medium yielded variants able to utilize hemoglobin and hemin suggesting alternate pathways for utilization of these two host iron sources. Lactoferrin utilization was dependent on malleobactin, but not pyochelin synthesis and/or uptake. A mba pch hmu hem quadruple mutant could use ferritin as an iron source and upon intranasal infection was lethal in an acute murine melioidosis model. These data suggest that B. pseudomallei may employ a novel ferritin-iron acquisition pathway as a means to sustain in vivo growth.

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Related in: MedlinePlus

Lethality of strain 1710b and its various iron acquisition mutants.A. Kaplan-Maier survival curves of BALB/c mice infected intranasally with 1710b (3×103 CFU), ΔMBA (3.5×103 CFU), Δ141-kb (2.1×103 CFU), Δ141-kb ΔfptA (1.9×103 CFU), or Δ141-kb ΔPCH ΔHMU ΔHEM (3.8×103 CFU). Survival was monitored and mice were euthanized upon reaching a pre-determined endpoint. Data were pooled from two independent experiments (total n = 10 per bacterial strain). For comparison of each mutant to 1710b the Bonferroni correction for multiple comparisons was applied. p values were as follows: ΔMBA, p = 0.12; Δ141-kb, p = 0.93; Δ141-kb ΔfptA, p = 0.0001; Δ141-kb ΔPCH ΔHMU ΔHEM, p = 0.19. B. Organ bacterial burden from endpoint mice (days 2.5 and 3) following intranasal challenge with 1710b or Δ141-kb ΔPCH ΔHMU ΔHEM. BALB/c mice (n = 10 per bacterial strain) were infected intranasally with 1710b (3×103 CFU) or Δ141-kb ΔPCH ΔHMU ΔHEM (3.8×103 CFU). Bacterial burdens from mice were determined in lung, liver and spleen using data pooled from mice euthanized at endpoint (days 2.5 and 3) as described in Materials and Methods. Significant differences between 1710b and Δ141-kb ΔPCH ΔHMU ΔHEM were determined by a two-tailed Student's t-test (*** = p<0.0001). Data are graphed as individual values, with bars representing the mean log10 CFU/organ titer for each group. Data were pooled from two independent experiments.
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pntd-0001715-g006: Lethality of strain 1710b and its various iron acquisition mutants.A. Kaplan-Maier survival curves of BALB/c mice infected intranasally with 1710b (3×103 CFU), ΔMBA (3.5×103 CFU), Δ141-kb (2.1×103 CFU), Δ141-kb ΔfptA (1.9×103 CFU), or Δ141-kb ΔPCH ΔHMU ΔHEM (3.8×103 CFU). Survival was monitored and mice were euthanized upon reaching a pre-determined endpoint. Data were pooled from two independent experiments (total n = 10 per bacterial strain). For comparison of each mutant to 1710b the Bonferroni correction for multiple comparisons was applied. p values were as follows: ΔMBA, p = 0.12; Δ141-kb, p = 0.93; Δ141-kb ΔfptA, p = 0.0001; Δ141-kb ΔPCH ΔHMU ΔHEM, p = 0.19. B. Organ bacterial burden from endpoint mice (days 2.5 and 3) following intranasal challenge with 1710b or Δ141-kb ΔPCH ΔHMU ΔHEM. BALB/c mice (n = 10 per bacterial strain) were infected intranasally with 1710b (3×103 CFU) or Δ141-kb ΔPCH ΔHMU ΔHEM (3.8×103 CFU). Bacterial burdens from mice were determined in lung, liver and spleen using data pooled from mice euthanized at endpoint (days 2.5 and 3) as described in Materials and Methods. Significant differences between 1710b and Δ141-kb ΔPCH ΔHMU ΔHEM were determined by a two-tailed Student's t-test (*** = p<0.0001). Data are graphed as individual values, with bars representing the mean log10 CFU/organ titer for each group. Data were pooled from two independent experiments.

Mentions: The lethality of 1710b and four of the 1710b-derived siderophore and hemin utilization mutants was tested in an acute intranasal (i.n.) challenge murine melioidosis model. BALB/c mice received a lethal i.n. challenge dose of LB-grown∼3×103 CFU of either strain 1710b or the mutants generated in this study which included ΔMBA, Δ141-kb, Δ141-kb ΔftpA, and Δ141-kb ΔPCH ΔHMU ΔHEM. Deletion of none of these genes or gene clusters significantly attenuated the lethality in the murine melioidosis model, with the quadruple Δ141-kb ΔPCH ΔHMU ΔHEMmutant showing a virtually indistinguishable survival curve when compared to the parental strain 1710b (Figure 6A). The types and timing of clinical symptoms development was similar following infection with individual strains and mice reached euthanasia endpoints at similar times (2.5 to 3.5 days). Although time-to-death was not significantly attenuated in the examined strains, dissemination was affected. Organ burdens with the quadruple iron acquisition mutant were significantly lower in the lung and spleen, but unchanged in the liver (Figure 6B). While we do not yet understand the differences in organ burdens with the various strains, it is interesting to note that Δ141-kb ΔPCH ΔHMU ΔHEM trends to multiply better in the liver, the primary organ for iron storage.


Burkholderia pseudomallei known siderophores and hemin uptake are dispensable for lethal murine melioidosis.

Kvitko BH, Goodyear A, Propst KL, Dow SW, Schweizer HP - PLoS Negl Trop Dis (2012)

Lethality of strain 1710b and its various iron acquisition mutants.A. Kaplan-Maier survival curves of BALB/c mice infected intranasally with 1710b (3×103 CFU), ΔMBA (3.5×103 CFU), Δ141-kb (2.1×103 CFU), Δ141-kb ΔfptA (1.9×103 CFU), or Δ141-kb ΔPCH ΔHMU ΔHEM (3.8×103 CFU). Survival was monitored and mice were euthanized upon reaching a pre-determined endpoint. Data were pooled from two independent experiments (total n = 10 per bacterial strain). For comparison of each mutant to 1710b the Bonferroni correction for multiple comparisons was applied. p values were as follows: ΔMBA, p = 0.12; Δ141-kb, p = 0.93; Δ141-kb ΔfptA, p = 0.0001; Δ141-kb ΔPCH ΔHMU ΔHEM, p = 0.19. B. Organ bacterial burden from endpoint mice (days 2.5 and 3) following intranasal challenge with 1710b or Δ141-kb ΔPCH ΔHMU ΔHEM. BALB/c mice (n = 10 per bacterial strain) were infected intranasally with 1710b (3×103 CFU) or Δ141-kb ΔPCH ΔHMU ΔHEM (3.8×103 CFU). Bacterial burdens from mice were determined in lung, liver and spleen using data pooled from mice euthanized at endpoint (days 2.5 and 3) as described in Materials and Methods. Significant differences between 1710b and Δ141-kb ΔPCH ΔHMU ΔHEM were determined by a two-tailed Student's t-test (*** = p<0.0001). Data are graphed as individual values, with bars representing the mean log10 CFU/organ titer for each group. Data were pooled from two independent experiments.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3383733&req=5

pntd-0001715-g006: Lethality of strain 1710b and its various iron acquisition mutants.A. Kaplan-Maier survival curves of BALB/c mice infected intranasally with 1710b (3×103 CFU), ΔMBA (3.5×103 CFU), Δ141-kb (2.1×103 CFU), Δ141-kb ΔfptA (1.9×103 CFU), or Δ141-kb ΔPCH ΔHMU ΔHEM (3.8×103 CFU). Survival was monitored and mice were euthanized upon reaching a pre-determined endpoint. Data were pooled from two independent experiments (total n = 10 per bacterial strain). For comparison of each mutant to 1710b the Bonferroni correction for multiple comparisons was applied. p values were as follows: ΔMBA, p = 0.12; Δ141-kb, p = 0.93; Δ141-kb ΔfptA, p = 0.0001; Δ141-kb ΔPCH ΔHMU ΔHEM, p = 0.19. B. Organ bacterial burden from endpoint mice (days 2.5 and 3) following intranasal challenge with 1710b or Δ141-kb ΔPCH ΔHMU ΔHEM. BALB/c mice (n = 10 per bacterial strain) were infected intranasally with 1710b (3×103 CFU) or Δ141-kb ΔPCH ΔHMU ΔHEM (3.8×103 CFU). Bacterial burdens from mice were determined in lung, liver and spleen using data pooled from mice euthanized at endpoint (days 2.5 and 3) as described in Materials and Methods. Significant differences between 1710b and Δ141-kb ΔPCH ΔHMU ΔHEM were determined by a two-tailed Student's t-test (*** = p<0.0001). Data are graphed as individual values, with bars representing the mean log10 CFU/organ titer for each group. Data were pooled from two independent experiments.
Mentions: The lethality of 1710b and four of the 1710b-derived siderophore and hemin utilization mutants was tested in an acute intranasal (i.n.) challenge murine melioidosis model. BALB/c mice received a lethal i.n. challenge dose of LB-grown∼3×103 CFU of either strain 1710b or the mutants generated in this study which included ΔMBA, Δ141-kb, Δ141-kb ΔftpA, and Δ141-kb ΔPCH ΔHMU ΔHEM. Deletion of none of these genes or gene clusters significantly attenuated the lethality in the murine melioidosis model, with the quadruple Δ141-kb ΔPCH ΔHMU ΔHEMmutant showing a virtually indistinguishable survival curve when compared to the parental strain 1710b (Figure 6A). The types and timing of clinical symptoms development was similar following infection with individual strains and mice reached euthanasia endpoints at similar times (2.5 to 3.5 days). Although time-to-death was not significantly attenuated in the examined strains, dissemination was affected. Organ burdens with the quadruple iron acquisition mutant were significantly lower in the lung and spleen, but unchanged in the liver (Figure 6B). While we do not yet understand the differences in organ burdens with the various strains, it is interesting to note that Δ141-kb ΔPCH ΔHMU ΔHEM trends to multiply better in the liver, the primary organ for iron storage.

Bottom Line: Prolonged incubation of a hmu hem mutant in hemoglobin-containing minimal medium yielded variants able to utilize hemoglobin and hemin suggesting alternate pathways for utilization of these two host iron sources.Lactoferrin utilization was dependent on malleobactin, but not pyochelin synthesis and/or uptake.These data suggest that B. pseudomallei may employ a novel ferritin-iron acquisition pathway as a means to sustain in vivo growth.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Immunology and Pathology, Rocky Mountain Regional Center of Excellence for Biodefense and Emerging Infectious Diseases Research, Colorado State University, Fort Collins, Colorado, United States of America.

ABSTRACT
Burkholderia pseudomallei is a mostly saprophytic bacterium, but can infect humans where it causes the difficult-to-manage disease melioidosis. Even with proper diagnosis and prompt therapeutic interventions mortality rates still range from >20% in Northern Australia to over 40% in Thailand. Surprisingly little is yet known about how B. pseudomallei infects, invades and survives within its hosts, and virtually nothing is known about the contribution of critical nutrients such as iron to the bacterium's pathogenesis. It was previously assumed that B. pseudomallei used iron-acquisition systems commonly found in other bacteria, for example siderophores. However, our previous discovery of a clinical isolate carrying a large chromosomal deletion missing the entire malleobactin gene cluster encoding the bacterium's major high-affinity siderophore while still being fully virulent in a murine melioidosis model suggested that other iron-acquisition systems might make contributions to virulence. Here, we deleted the major siderophore malleobactin (mba) and pyochelin (pch) gene clusters in strain 1710b and revealed a residual siderophore activity which was unrelated to other known Burkholderia siderophores such as cepabactin and cepaciachelin, and not due to increased secretion of chelators such as citrate. Deletion of the two hemin uptake loci, hmu and hem, showed that Hmu is required for utilization of hemin and hemoglobin and that Hem cannot complement a Hmu deficiency. Prolonged incubation of a hmu hem mutant in hemoglobin-containing minimal medium yielded variants able to utilize hemoglobin and hemin suggesting alternate pathways for utilization of these two host iron sources. Lactoferrin utilization was dependent on malleobactin, but not pyochelin synthesis and/or uptake. A mba pch hmu hem quadruple mutant could use ferritin as an iron source and upon intranasal infection was lethal in an acute murine melioidosis model. These data suggest that B. pseudomallei may employ a novel ferritin-iron acquisition pathway as a means to sustain in vivo growth.

Show MeSH
Related in: MedlinePlus