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Identification of a predicted trimeric autotransporter adhesin required for biofilm formation of Burkholderia pseudomallei.

Lazar Adler NR, Dean RE, Saint RJ, Stevens MP, Prior JL, Atkins TP, Galyov EE - PLoS ONE (2013)

Bottom Line: The bpss1439 mutant demonstrated a significant reduction in biofilm formation at 48 hours in comparison to its parent 10276 wild-type strain.Additionally, it was observed that this phenotype was due to low levels of bacterial adhesion to the abiotic surface as well as reduced microcolony formation.Taken together, these studies indicate that BPSS1439 is a novel predicted autotransporter involved in biofilm formation of B. pseudomallei; hence, this factor was named BbfA, Burkholderia biofilm factor A.

View Article: PubMed Central - PubMed

Affiliation: Department of Infection, Immunity and Inflammation, University of Leicester, Leicester, United Kingdom.

ABSTRACT
The autotransporters are a large and diverse family of bacterial secreted and outer membrane proteins, which are present in many Gram-negative bacterial pathogens and play a role in numerous environmental and virulence-associated interactions. As part of a larger systematic study on the autotransporters of Burkholderia pseudomallei, the causative agent of the severe tropical disease melioidosis, we have constructed an insertion mutant in the bpss1439 gene encoding an unstudied predicted trimeric autotransporter adhesin. The bpss1439 mutant demonstrated a significant reduction in biofilm formation at 48 hours in comparison to its parent 10276 wild-type strain. This phenotype was complemented to wild-type levels by the introduction of a full-length copy of the bpss1439 gene in trans. Examination of the wild-type and bpss1439 mutant strains under biofilm-inducing conditions by microscopy after 48 hours confirmed that the bpss1439 mutant produced less biofilm compared to wild-type. Additionally, it was observed that this phenotype was due to low levels of bacterial adhesion to the abiotic surface as well as reduced microcolony formation. In a murine melioidosis model, the bpss1439 mutant strain demonstrated a moderate attenuation for virulence compared to the wild-type strain. This attenuation was abrogated by in trans complementation, suggesting that bpss1439 plays a subtle role in the pathogenesis of B. pseudomallei. Taken together, these studies indicate that BPSS1439 is a novel predicted autotransporter involved in biofilm formation of B. pseudomallei; hence, this factor was named BbfA, Burkholderia biofilm factor A.

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Mice infected with the bbfA mutant demonstrated a delay to death in comparison to those challenged with the parental strain.Groups of six BALB/c mice were challenged via the intra-peritoneal route and the median time to death was determined by the Mantel-Cox log-rank test at 35 days post-infection. Mice inoculated with the bbfA mutant had a mean survival time of 3.5 days versus 2 days for those dosed with the wild-type (p = 0.03).
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pone-0079461-g004: Mice infected with the bbfA mutant demonstrated a delay to death in comparison to those challenged with the parental strain.Groups of six BALB/c mice were challenged via the intra-peritoneal route and the median time to death was determined by the Mantel-Cox log-rank test at 35 days post-infection. Mice inoculated with the bbfA mutant had a mean survival time of 3.5 days versus 2 days for those dosed with the wild-type (p = 0.03).

Mentions: To assess the role of biofilm formation in the pathogenesis of B. pseudomallei, the bbfA mutant was tested for virulence in a murine melioidosis model. Groups of six BALB/c mice were challenged via the intra-peritoneal route and median time to death was determined by the Mantel-Cox log-rank test at 35 days post-infection (Figure 4). Compared to mice infected with the parent 10276 wild-type, the mice challenged with the bbfA mutant demonstrated a significant delay in time to death, with a mean survival time of 3.5 days versus 2 days for the wild-type (p = 0.03). However, this delay in time to death could, in part, be a product of the marginally lower inoculum (3.07×105 CFU for bbfA vs. 3.5×105 CFU for the wild-type); therefore, subsequent animal studies were performed. Six groups of five BALB/c mice were challenged via the intra-peritoneal route and the median lethal dose (mice were humanely culled when pre-defined end-points were reached) was determined by the using the Reed and Meunch calculation based on cumulative lethal dose. Compared to the parent 10276 wild-type, the bbfA mutant demonstrated a 13-fold increase in medium lethal dose (2.09×105 vs. 1.6×104 CFU). To confirm that this attenuation is directly linked to BbfA, in trans complementation of the bbfA mutant was performed using a competitive in vivo growth assay. An equal ratio of the wild-type harbouring empty pME6032 vector and the bbfA mutant harbouring either pME6032 or the pME-1439 complementation vector was inoculated into groups of three BALB/c mice via the intra-peritoneal route. Mice were humanely culled at 24 hours post-infection and livers and spleens were harvested and plated for bacterial recovery. Colonies were patched to determine the percentage of in vivo growth by the bbfA mutant versus the wild-type strain by subtracting the number of tetR, kanR (bbfA mutant harbouring pME6032 or pME-1439) colonies from the number of tetR, kanS (wild-type harbouring pME6032) colonies. The bbfA mutant harbouring empty pME6032 vector demonstrated reduced in vivo growth compared to the wild-type harbouring pME6032. However, this attenuation was abrogated by the complemented bbfA mutant strain that displayed a comparable level of in vivo survival to the wild-type harbouring pME6032, both within the spleen (0.33%±0.58% vs. 42.3%±12.3%, p = 0.004) and the liver (0%±0% vs. 42.5%±20.6%, p = 0.023). Bacteria isolated from the liver and spleen of control mice infected with only the complemented bbfA mutant strain demonstrated between 0% to 4% loss of the pME-1439 plasmid over the 24 hour experimental period. These data suggest that bbfA-mediated biofilm formation may play a subtle role in the pathogenesis of melioidosis. Future studies could focus on whether biofilm production relates to chronic disease rather than the acute infection seen in the BALB/c melioidosis model [6], or when mice are challenged at a mucosal surface rather than by a parenteral route. Alternatively, studies could examine differences between strains; it has been previously observed that B. pseudomallei strains demonstrate varying levels of biofilm production, while B. mallei and B. thailandensis strains produce relatively low levels of biofilm [4], [6], [7]. Therefore, it is of interest to note that the bbfA gene occurs as a pseudogene in some B. pseudomallei strains, as well as B. thailandensis and B. mallei, and this may explain the variable biofilm levels seen.


Identification of a predicted trimeric autotransporter adhesin required for biofilm formation of Burkholderia pseudomallei.

Lazar Adler NR, Dean RE, Saint RJ, Stevens MP, Prior JL, Atkins TP, Galyov EE - PLoS ONE (2013)

Mice infected with the bbfA mutant demonstrated a delay to death in comparison to those challenged with the parental strain.Groups of six BALB/c mice were challenged via the intra-peritoneal route and the median time to death was determined by the Mantel-Cox log-rank test at 35 days post-infection. Mice inoculated with the bbfA mutant had a mean survival time of 3.5 days versus 2 days for those dosed with the wild-type (p = 0.03).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3818227&req=5

pone-0079461-g004: Mice infected with the bbfA mutant demonstrated a delay to death in comparison to those challenged with the parental strain.Groups of six BALB/c mice were challenged via the intra-peritoneal route and the median time to death was determined by the Mantel-Cox log-rank test at 35 days post-infection. Mice inoculated with the bbfA mutant had a mean survival time of 3.5 days versus 2 days for those dosed with the wild-type (p = 0.03).
Mentions: To assess the role of biofilm formation in the pathogenesis of B. pseudomallei, the bbfA mutant was tested for virulence in a murine melioidosis model. Groups of six BALB/c mice were challenged via the intra-peritoneal route and median time to death was determined by the Mantel-Cox log-rank test at 35 days post-infection (Figure 4). Compared to mice infected with the parent 10276 wild-type, the mice challenged with the bbfA mutant demonstrated a significant delay in time to death, with a mean survival time of 3.5 days versus 2 days for the wild-type (p = 0.03). However, this delay in time to death could, in part, be a product of the marginally lower inoculum (3.07×105 CFU for bbfA vs. 3.5×105 CFU for the wild-type); therefore, subsequent animal studies were performed. Six groups of five BALB/c mice were challenged via the intra-peritoneal route and the median lethal dose (mice were humanely culled when pre-defined end-points were reached) was determined by the using the Reed and Meunch calculation based on cumulative lethal dose. Compared to the parent 10276 wild-type, the bbfA mutant demonstrated a 13-fold increase in medium lethal dose (2.09×105 vs. 1.6×104 CFU). To confirm that this attenuation is directly linked to BbfA, in trans complementation of the bbfA mutant was performed using a competitive in vivo growth assay. An equal ratio of the wild-type harbouring empty pME6032 vector and the bbfA mutant harbouring either pME6032 or the pME-1439 complementation vector was inoculated into groups of three BALB/c mice via the intra-peritoneal route. Mice were humanely culled at 24 hours post-infection and livers and spleens were harvested and plated for bacterial recovery. Colonies were patched to determine the percentage of in vivo growth by the bbfA mutant versus the wild-type strain by subtracting the number of tetR, kanR (bbfA mutant harbouring pME6032 or pME-1439) colonies from the number of tetR, kanS (wild-type harbouring pME6032) colonies. The bbfA mutant harbouring empty pME6032 vector demonstrated reduced in vivo growth compared to the wild-type harbouring pME6032. However, this attenuation was abrogated by the complemented bbfA mutant strain that displayed a comparable level of in vivo survival to the wild-type harbouring pME6032, both within the spleen (0.33%±0.58% vs. 42.3%±12.3%, p = 0.004) and the liver (0%±0% vs. 42.5%±20.6%, p = 0.023). Bacteria isolated from the liver and spleen of control mice infected with only the complemented bbfA mutant strain demonstrated between 0% to 4% loss of the pME-1439 plasmid over the 24 hour experimental period. These data suggest that bbfA-mediated biofilm formation may play a subtle role in the pathogenesis of melioidosis. Future studies could focus on whether biofilm production relates to chronic disease rather than the acute infection seen in the BALB/c melioidosis model [6], or when mice are challenged at a mucosal surface rather than by a parenteral route. Alternatively, studies could examine differences between strains; it has been previously observed that B. pseudomallei strains demonstrate varying levels of biofilm production, while B. mallei and B. thailandensis strains produce relatively low levels of biofilm [4], [6], [7]. Therefore, it is of interest to note that the bbfA gene occurs as a pseudogene in some B. pseudomallei strains, as well as B. thailandensis and B. mallei, and this may explain the variable biofilm levels seen.

Bottom Line: The bpss1439 mutant demonstrated a significant reduction in biofilm formation at 48 hours in comparison to its parent 10276 wild-type strain.Additionally, it was observed that this phenotype was due to low levels of bacterial adhesion to the abiotic surface as well as reduced microcolony formation.Taken together, these studies indicate that BPSS1439 is a novel predicted autotransporter involved in biofilm formation of B. pseudomallei; hence, this factor was named BbfA, Burkholderia biofilm factor A.

View Article: PubMed Central - PubMed

Affiliation: Department of Infection, Immunity and Inflammation, University of Leicester, Leicester, United Kingdom.

ABSTRACT
The autotransporters are a large and diverse family of bacterial secreted and outer membrane proteins, which are present in many Gram-negative bacterial pathogens and play a role in numerous environmental and virulence-associated interactions. As part of a larger systematic study on the autotransporters of Burkholderia pseudomallei, the causative agent of the severe tropical disease melioidosis, we have constructed an insertion mutant in the bpss1439 gene encoding an unstudied predicted trimeric autotransporter adhesin. The bpss1439 mutant demonstrated a significant reduction in biofilm formation at 48 hours in comparison to its parent 10276 wild-type strain. This phenotype was complemented to wild-type levels by the introduction of a full-length copy of the bpss1439 gene in trans. Examination of the wild-type and bpss1439 mutant strains under biofilm-inducing conditions by microscopy after 48 hours confirmed that the bpss1439 mutant produced less biofilm compared to wild-type. Additionally, it was observed that this phenotype was due to low levels of bacterial adhesion to the abiotic surface as well as reduced microcolony formation. In a murine melioidosis model, the bpss1439 mutant strain demonstrated a moderate attenuation for virulence compared to the wild-type strain. This attenuation was abrogated by in trans complementation, suggesting that bpss1439 plays a subtle role in the pathogenesis of B. pseudomallei. Taken together, these studies indicate that BPSS1439 is a novel predicted autotransporter involved in biofilm formation of B. pseudomallei; hence, this factor was named BbfA, Burkholderia biofilm factor A.

Show MeSH
Related in: MedlinePlus