Crystal structure of <scp><i>B</i></scp><i>acillus anthracis</i> virulence regulator <scp>AtxA</scp> and effects of phosphorylated histidines on multimerization and activity

Hammerstrom, Troy G.; Banks, Lori; Swick, Michelle C.; Joachimiak, A.; Osipiuk, J.; Koehler, Theresa M.

doi:10.1111/mmi.12867

Cited by 32 publications

(68 citation statements)

References 71 publications

(122 reference statements)

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“…The current hypothesis is that, while these proteins have the ability to dimerize, it is the ability of the dimers to come together to form higher-order oligomers that enables them to regulate gene expression. Consistent with this are recent findings that Mga and AtxA can be phosphorylated to modulate their activity and that this is due not to modification of DNA binding but rather to modification of the ability of these proteins to form multimers (25,26,34). Our data here are consistent with the hypothesis that multimerization plays an important role in controlling the activity of Mga-like regulators.…”

Section: Discussionsupporting

confidence: 81%

“…RivR is a member of the Mga-like family of transcriptional regulators, which includes the GAS protein Mga and the B. anthracis protein AtxA. Recently, it was shown that both Mga and AtxA form multimers and that this function is required for activity (22,(24)(25)(26). Therefore, we wished to determine whether RivR also formed multimers and, if so, what degree of multimers were formed.…”

Section: Resultsmentioning

confidence: 99%

“…For example, phosphotransferase system components carry out the phosphorylation of histidine residues within the PRDs of Mga and AtxA, linking regulatory activity to carbohydrate metabolism (25,26,34). Whether any of the 13 histidine residues located within the PRDs of RivR are modified in a similar manner, and the functional consequences of such modification, remain to be determined.…”

Section: Discussionmentioning

confidence: 99%

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Multimerization of the Virulence-Enhancing Group A Streptococcus Transcription Factor RivR Is Required for Regulatory Activity

et al. 2017

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Group A Streptococcus (GAS) (Streptococcus pyogenes) causes more than 700 million human infections each year. The significant morbidity and mortality rates associated with GAS infections are in part a consequence of the ability of this pathogen to coordinately regulate virulence factor expression during infection. RofA-like protein IV (RivR) is a member of the Mga-like family of transcriptional regulators, and previously we reported that RivR negatively regulates transcription of the hasA and grab virulence factor-encoding genes. Here, we determined that RivR inhibits the ability of GAS to survive and to replicate in human blood. To begin to assess the biochemical basis of RivR activity, we investigated its ability to form multimers, which is a characteristic of Mga-like proteins. We found that RivR forms both dimers and a higher-molecular-mass multimer, which we hypothesize is a tetramer. As cysteine residues are known to contribute to the ability of proteins to dimerize, we created a library of expression plasmids in which each of the four cysteines in RivR was converted to serine. While the C68S RivR protein was essentially unaffected in its ability to dimerize, the C32S and C377S proteins were attenuated, while the C470S protein completely lacked the ability to dimerize. Consistent with dimerization being required for regulatory activity, the C470S RivR protein was unable to repress hasA and grab gene expression in a rivR mutant. Thus, multimer formation is a prerequisite for RivR activity, which supports recent data obtained for other Mga-like family members, suggesting a common regulatory mechanism.IMPORTANCE The modulation of gene transcription is key to the ability of bacterial pathogens to infect hosts to cause disease. Here, we discovered that the group A Streptococcus transcription factor RivR negatively regulates the ability of this pathogen to survive in human blood, and we also began biochemical characterization of this protein. We determined that, in order for RivR to function, it must self-associate, forming both dimers (consisting of two RivR proteins) and higher-order complexes (consisting of more than two RivR proteins). This functional requirement for RivR is shared by other regulators in the same family of proteins, suggesting a common regulatory mechanism. Insight into how these transcription factors function may facilitate the development of novel therapeutic agents targeting their activity.KEYWORDS multimer, transcription factor, virulence regulation, Streptococcus pyogenes T he human bacterial pathogen group A Streptococcus (GAS) causes a diverse range of infections, from the superficial, such as pharyngitis, to the severely invasive, such as necrotizing fasciitis (1-3). In addition, failure to adequately treat individuals with skin

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Section: Discussionsupporting

confidence: 81%

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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Multimerization of the Virulence-Enhancing Group A Streptococcus Transcription Factor RivR Is Required for Regulatory Activity

et al. 2017

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“…To deduce the molecular mechanism of gene regulation by Mga, we first performed modeling studies with I-TASSER (30)(31)(32). Mga is predicted to share a high degree of structural homology with AtxA, a PRD-containing activator from Bacillus anthracis (PDB accession number 4R6I) (10,33,34). The predicted model has a high template modeling (TM) score of 0.62 Ϯ 0.14 (a score of Ͼ0.5 is reliable), suggesting that the model has the correct topology (30)(31)(32).…”

Section: Resultsmentioning

confidence: 99%

Phosphorylation Events in the Multiple Gene Regulator of Group A Streptococcus Significantly Influence Global Gene Expression and Virulence

et al. 2015

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bWhole-genome sequencing analysis of ϳ800 strains of group A Streptococcus (GAS) found that the gene encoding the multiple virulence gene regulator of GAS (mga) is highly polymorphic in serotype M59 strains but not in strains of other serotypes. To help understand the molecular mechanism of gene regulation by Mga and its contribution to GAS pathogenesis in serotype M59 GAS, we constructed an isogenic mga mutant strain. Transcriptome studies indicated a significant regulatory influence of Mga and altered metabolic capabilities conferred by Mga-regulated genes. We assessed the phosphorylation status of Mga in GAS cell lysates with Phos-tag gels. The results revealed that Mga is phosphorylated at histidines in vivo. Using phosphomimetic and nonphosphomimetic substitutions at conserved phosphoenolpyruvate:carbohydrate phosphotransferase regulation domain (PRD) histidines of Mga, we demonstrated that phosphorylation-mimicking aspartate replacements at H207 and H273 of PRD-1 and at H327 of PRD-2 are inhibitory to Mga-dependent gene expression. Conversely, non-phosphorylation-mimicking alanine substitutions at H273 and H327 relieved inhibition, and the mutant strains exhibited a wild-type phenotype. The opposing regulatory profiles observed for phosphorylation-and non-phosphorylation-mimicking substitutions at H273 extended to global gene regulation by Mga. Consistent with these observations, the H273D mutant strain attenuated GAS virulence, whereas the H273A strain exhibited a wild-type virulence phenotype in a mouse model of necrotizing fasciitis. Together, our results demonstrate phosphoregulation of Mga and its direct link to virulence in M59 GAS strains. These data also lay a foundation toward understanding how naturally occurring gain-of-function variations in mga, such as H201R, may confer an advantage to the pathogen and contribute to M59 GAS pathogenesis.

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“…For example, in Bacillus anthracis, the causative agent of the zoonotic infectious disease, anthrax, the plasmid-borne atxA gene encodes a transcription factor required for virulence, since it is necessary for the activation of genes for toxin and protective capsule production (5,6). Other Grampositive bacteria possess genes encoding one or more forms of the ArsC (arsenate reductase) family protein, Spx, which is an RNA polymerase (RNAP)-binding protein directing the expression of large regulons that include genes necessary for pathogenesis (7)(8)(9)(10)(11).…”

mentioning

confidence: 99%

Evidence that Oxidative Stress Induces spxA2 Transcription in Bacillus anthracis Sterne through a Mechanism Requiring SpxA1 and Positive Autoregulation

et al. 2016

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Bacillus anthracis possesses two paralogs of the transcriptional regulator, Spx. SpxA1 and SpxA2 interact with RNA polymerase (RNAP) to activate the transcription of genes implicated in the prevention and alleviation of oxidative protein damage. The spxA2 gene is highly upregulated in infected macrophages, but how this is achieved is unknown. Previous studies have shown that the spxA2 gene was under negative control by the Rrf2 family repressor protein, SaiR, whose activity is sensitive to oxidative stress. These studies also suggested that spxA2 was under positive autoregulation. In the present study, we show by in vivo and in vitro analyses that spxA2 is under direct autoregulation but is also dependent on the SpxA1 paralogous protein. The deletion of either spxA1 or spxA2 reduced the diamide-inducible expression of an spxA2-lacZ construct. In vitro transcription reactions using purified B. anthracis RNAP showed that SpxA1 and SpxA2 protein stimulates transcription from a DNA fragment containing the spxA2 promoter. Ectopically positioned spxA2-lacZ fusion requires both SpxA1 and SpxA2 for expression, but the requirement for SpxA1 is partially overcome when saiR is deleted. Electrophoretic mobility shift assays showed that SpxA1 and SpxA2 enhance the affinity of RNAP for spxA2 promoter DNA and that this activity is sensitive to reductant. We hypothesize that the previously observed upregulation of spxA2 in the oxidative environment of the macrophage is at least partly due to SpxA1-mediated SaiR repressor inactivation and the positive autoregulation of spxA2 transcription. T ranscription factors of pathogenic bacteria can be important virulence determinants, since they control genes that specify toxins, proteins functioning in nutrient acquisition within the hostile host environment, and products that operate by subverting the host innate immunity (1-4). For example, in Bacillus anthracis, the causative agent of the zoonotic infectious disease, anthrax, the plasmid-borne atxA gene encodes a transcription factor required for virulence, since it is necessary for the activation of genes for toxin and protective capsule production (5, 6). Other Grampositive bacteria possess genes encoding one or more forms of the ArsC (arsenate reductase) family protein, Spx, which is an RNA polymerase (RNAP)-binding protein directing the expression of large regulons that include genes necessary for pathogenesis (7-11). Studies of streptococci, Listeria monocytogenes, Staphylococcus aureus, and Enterococcus spp. have demonstrated the requirement for Spx proteins for virulence and viability (7,8,(12)(13)(14)(15). IMPORTANCE Regulators of transcription initiationSpx was detected in studies of B. subtilis, in which it is encoded by a gene that is under complex transcriptional control involving four RNAP forms and two repressors (16)(17)(18)(19). The complex control of spx transcription operates in response to various stress conditions, such as oxidative and cell envelope stress. Spx protein levels increase under conditions caus...

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Crystal structure of Bacillus anthracis virulence regulator AtxA and effects of phosphorylated histidines on multimerization and activity

Cited by 32 publications

References 71 publications

Multimerization of the Virulence-Enhancing Group A Streptococcus Transcription Factor RivR Is Required for Regulatory Activity

Multimerization of the Virulence-Enhancing Group A Streptococcus Transcription Factor RivR Is Required for Regulatory Activity

Phosphorylation Events in the Multiple Gene Regulator of Group A Streptococcus Significantly Influence Global Gene Expression and Virulence

Evidence that Oxidative Stress Induces spxA2 Transcription in Bacillus anthracis Sterne through a Mechanism Requiring SpxA1 and Positive Autoregulation

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