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The arginine-ornithine antiporter ArcD contributes to biological fitness of Streptococcus suis.

Fulde M, Willenborg J, Huber C, Hitzmann A, Willms D, Seitz M, Eisenreich W, Valentin-Weigand P, Goethe R - Front Cell Infect Microbiol (2014)

Bottom Line: The arginine-ornithine antiporter (ArcD) is part of the Arginine Deiminase System (ADS), a catabolic, energy-providing pathway found in a variety of different bacterial species, including the porcine zoonotic pathogen Streptococcus suis.In the present study, we showed by experiments using [U-(13)C6]arginine as a tracer molecule that S. suis is auxotrophic for arginine and that bacterial growth depends on the uptake of extracellular arginine.Based on these results, we propose that ArcD, by its function as an arginine-ornithine antiporter, is important for supplying arginine as substrate of the ADS and, thereby, contributes to biological fitness and virulence of S. suis in the host.

View Article: PubMed Central - PubMed

Affiliation: Department of Infectious Diseases, Institute for Microbiology, University of Veterinary Medicine Hannover, Germany ; Department of Medical Microbiology, Helmholtz Centre for Infection Research (HZI) Braunschweig, Germany.

ABSTRACT
The arginine-ornithine antiporter (ArcD) is part of the Arginine Deiminase System (ADS), a catabolic, energy-providing pathway found in a variety of different bacterial species, including the porcine zoonotic pathogen Streptococcus suis. The ADS has recently been shown to play a role in the pathogenicity of S. suis, in particular in its survival in host cells. The contribution of arginine and arginine transport mediated by ArcD, however, has yet to be clarified. In the present study, we showed by experiments using [U-(13)C6]arginine as a tracer molecule that S. suis is auxotrophic for arginine and that bacterial growth depends on the uptake of extracellular arginine. To further study the role of ArcD in arginine metabolism, we generated an arcD-specific mutant strain and characterized its growth compared to the wild-type (WT) strain, a virulent serotype 2 strain. The mutant strain showed a markedly reduced growth in chemically defined media supplemented with arginine when compared to the WT strain, suggesting that ArcD promotes arginine uptake. To further evaluate the in vivo relevance of ArcD, we studied the intracellular bacterial survival of the arcD mutant strain in an epithelial cell culture infection model. The mutant strain was substantially attenuated, and its reduced intracellular survival rate correlated with a lower ability to neutralize the acidified environment. Based on these results, we propose that ArcD, by its function as an arginine-ornithine antiporter, is important for supplying arginine as substrate of the ADS and, thereby, contributes to biological fitness and virulence of S. suis in the host.

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The S. suis ArcD contributes to environmental pH homeostasis and biological fitness in an arginine-dependent manner. (A) Growth kinetics of S. suis WT strain 10 (red) and its isogenic arcD-deficient mutant strain 10ΔarcD (blue) in a tryptone-based medium containing 10 mM galactose (dotted lines) and 50 mM arginine (solid lines) if indicated. The optical density at 600 nm OD600 was measured every hour. Data represent means and standard deviations of a representative experiment performed in triplicates. Experiments were repeated at least two times. (B) Bacteria were grown as described in (A), the course of pH values were measured every hour. Data represent means and standard deviations of a representative experiment performed in triplicates. Statistics were performed in a one-tailed t-test with p < 0.01. (C) Ammonia production of the culture supernatant was measured after 24 h of growth. Results are given as mg ammonia per ml of medium (ml−1). Statistical significance is indicated for a one-sided t-test (***p < 0.001; ns, not significant). (D) The amount of arginine in the culture supernatant of WT strain 10 (red bar) and 10ΔarcD (blue) was detected by a method adapted from Sakaguchi (1925). Results are given in mM arginine. TY medium alone (white bar) served as negative control.
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Figure 2: The S. suis ArcD contributes to environmental pH homeostasis and biological fitness in an arginine-dependent manner. (A) Growth kinetics of S. suis WT strain 10 (red) and its isogenic arcD-deficient mutant strain 10ΔarcD (blue) in a tryptone-based medium containing 10 mM galactose (dotted lines) and 50 mM arginine (solid lines) if indicated. The optical density at 600 nm OD600 was measured every hour. Data represent means and standard deviations of a representative experiment performed in triplicates. Experiments were repeated at least two times. (B) Bacteria were grown as described in (A), the course of pH values were measured every hour. Data represent means and standard deviations of a representative experiment performed in triplicates. Statistics were performed in a one-tailed t-test with p < 0.01. (C) Ammonia production of the culture supernatant was measured after 24 h of growth. Results are given as mg ammonia per ml of medium (ml−1). Statistical significance is indicated for a one-sided t-test (***p < 0.001; ns, not significant). (D) The amount of arginine in the culture supernatant of WT strain 10 (red bar) and 10ΔarcD (blue) was detected by a method adapted from Sakaguchi (1925). Results are given in mM arginine. TY medium alone (white bar) served as negative control.

Mentions: In silico analysis of the S. suis ArcD revealed significant homologies to transmembranal proteins with arginine-ornithine antiporter function of other streptococci and other arginine-fermenting bacteria (Table 1). However, functional studies on this topic are rare. Therefore, we inactivated arcD by insertion mutagenesis and characterized the phenotype of the mutant by growth kinetics. As depicted in Figure 2A, a comparable growth of WT strain 10 and its isogenic mutant strain 10ΔarcD was observed in the first hours of growth with a mean OD600 ranging between 0.0279 ± 0.008 (WT, with arginine supplementation) and 0.0315 ± 0.0055 (10ΔarcD, without arginine supplementation). After 4 h, the growth of WT strain 10 (red lines) was higher as compared to the arcD-deficient mutant strain (blue lines). Interestingly, supplementation of arginine (solid lines) did not lead to a higher growth, neither of WT strain 10 nor of the mutant until 5 h. After 6 h, WT strain 10 reached an OD600 of 0.2253 ± 0.047 (broken red line) without arginine supplementation, and an OD600 of 0.3983 ± 0.12 (solid red line) when arginine had been supplemented. In contrast, significantly lower OD values were detected for the arcD-deficient mutant strain, with (0.1343 ± 0.025, solid blue line) and without arginine supplementation (0.093 ± 0.001, broken blue line). Nevertheless, though less prominent the mutant strain 10ΔarcD showed an arginine-dependent phenotype similar to the WT strain. Overall, differences in bacterial numbers and arginine availability increased over time between both strains.


The arginine-ornithine antiporter ArcD contributes to biological fitness of Streptococcus suis.

Fulde M, Willenborg J, Huber C, Hitzmann A, Willms D, Seitz M, Eisenreich W, Valentin-Weigand P, Goethe R - Front Cell Infect Microbiol (2014)

The S. suis ArcD contributes to environmental pH homeostasis and biological fitness in an arginine-dependent manner. (A) Growth kinetics of S. suis WT strain 10 (red) and its isogenic arcD-deficient mutant strain 10ΔarcD (blue) in a tryptone-based medium containing 10 mM galactose (dotted lines) and 50 mM arginine (solid lines) if indicated. The optical density at 600 nm OD600 was measured every hour. Data represent means and standard deviations of a representative experiment performed in triplicates. Experiments were repeated at least two times. (B) Bacteria were grown as described in (A), the course of pH values were measured every hour. Data represent means and standard deviations of a representative experiment performed in triplicates. Statistics were performed in a one-tailed t-test with p < 0.01. (C) Ammonia production of the culture supernatant was measured after 24 h of growth. Results are given as mg ammonia per ml of medium (ml−1). Statistical significance is indicated for a one-sided t-test (***p < 0.001; ns, not significant). (D) The amount of arginine in the culture supernatant of WT strain 10 (red bar) and 10ΔarcD (blue) was detected by a method adapted from Sakaguchi (1925). Results are given in mM arginine. TY medium alone (white bar) served as negative control.
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Related In: Results  -  Collection

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Figure 2: The S. suis ArcD contributes to environmental pH homeostasis and biological fitness in an arginine-dependent manner. (A) Growth kinetics of S. suis WT strain 10 (red) and its isogenic arcD-deficient mutant strain 10ΔarcD (blue) in a tryptone-based medium containing 10 mM galactose (dotted lines) and 50 mM arginine (solid lines) if indicated. The optical density at 600 nm OD600 was measured every hour. Data represent means and standard deviations of a representative experiment performed in triplicates. Experiments were repeated at least two times. (B) Bacteria were grown as described in (A), the course of pH values were measured every hour. Data represent means and standard deviations of a representative experiment performed in triplicates. Statistics were performed in a one-tailed t-test with p < 0.01. (C) Ammonia production of the culture supernatant was measured after 24 h of growth. Results are given as mg ammonia per ml of medium (ml−1). Statistical significance is indicated for a one-sided t-test (***p < 0.001; ns, not significant). (D) The amount of arginine in the culture supernatant of WT strain 10 (red bar) and 10ΔarcD (blue) was detected by a method adapted from Sakaguchi (1925). Results are given in mM arginine. TY medium alone (white bar) served as negative control.
Mentions: In silico analysis of the S. suis ArcD revealed significant homologies to transmembranal proteins with arginine-ornithine antiporter function of other streptococci and other arginine-fermenting bacteria (Table 1). However, functional studies on this topic are rare. Therefore, we inactivated arcD by insertion mutagenesis and characterized the phenotype of the mutant by growth kinetics. As depicted in Figure 2A, a comparable growth of WT strain 10 and its isogenic mutant strain 10ΔarcD was observed in the first hours of growth with a mean OD600 ranging between 0.0279 ± 0.008 (WT, with arginine supplementation) and 0.0315 ± 0.0055 (10ΔarcD, without arginine supplementation). After 4 h, the growth of WT strain 10 (red lines) was higher as compared to the arcD-deficient mutant strain (blue lines). Interestingly, supplementation of arginine (solid lines) did not lead to a higher growth, neither of WT strain 10 nor of the mutant until 5 h. After 6 h, WT strain 10 reached an OD600 of 0.2253 ± 0.047 (broken red line) without arginine supplementation, and an OD600 of 0.3983 ± 0.12 (solid red line) when arginine had been supplemented. In contrast, significantly lower OD values were detected for the arcD-deficient mutant strain, with (0.1343 ± 0.025, solid blue line) and without arginine supplementation (0.093 ± 0.001, broken blue line). Nevertheless, though less prominent the mutant strain 10ΔarcD showed an arginine-dependent phenotype similar to the WT strain. Overall, differences in bacterial numbers and arginine availability increased over time between both strains.

Bottom Line: The arginine-ornithine antiporter (ArcD) is part of the Arginine Deiminase System (ADS), a catabolic, energy-providing pathway found in a variety of different bacterial species, including the porcine zoonotic pathogen Streptococcus suis.In the present study, we showed by experiments using [U-(13)C6]arginine as a tracer molecule that S. suis is auxotrophic for arginine and that bacterial growth depends on the uptake of extracellular arginine.Based on these results, we propose that ArcD, by its function as an arginine-ornithine antiporter, is important for supplying arginine as substrate of the ADS and, thereby, contributes to biological fitness and virulence of S. suis in the host.

View Article: PubMed Central - PubMed

Affiliation: Department of Infectious Diseases, Institute for Microbiology, University of Veterinary Medicine Hannover, Germany ; Department of Medical Microbiology, Helmholtz Centre for Infection Research (HZI) Braunschweig, Germany.

ABSTRACT
The arginine-ornithine antiporter (ArcD) is part of the Arginine Deiminase System (ADS), a catabolic, energy-providing pathway found in a variety of different bacterial species, including the porcine zoonotic pathogen Streptococcus suis. The ADS has recently been shown to play a role in the pathogenicity of S. suis, in particular in its survival in host cells. The contribution of arginine and arginine transport mediated by ArcD, however, has yet to be clarified. In the present study, we showed by experiments using [U-(13)C6]arginine as a tracer molecule that S. suis is auxotrophic for arginine and that bacterial growth depends on the uptake of extracellular arginine. To further study the role of ArcD in arginine metabolism, we generated an arcD-specific mutant strain and characterized its growth compared to the wild-type (WT) strain, a virulent serotype 2 strain. The mutant strain showed a markedly reduced growth in chemically defined media supplemented with arginine when compared to the WT strain, suggesting that ArcD promotes arginine uptake. To further evaluate the in vivo relevance of ArcD, we studied the intracellular bacterial survival of the arcD mutant strain in an epithelial cell culture infection model. The mutant strain was substantially attenuated, and its reduced intracellular survival rate correlated with a lower ability to neutralize the acidified environment. Based on these results, we propose that ArcD, by its function as an arginine-ornithine antiporter, is important for supplying arginine as substrate of the ADS and, thereby, contributes to biological fitness and virulence of S. suis in the host.

Show MeSH
Related in: MedlinePlus