Kellie Burnside - Academia.edu (original) (raw)

Papers by Kellie Burnside

Journal of Bacteriology, 2003

Molecular Microbiology, May 1, 2010

The pathogen encodes a number of virulence factors including the pluripotent b-haemolysin/cytolys... more The pathogen encodes a number of virulence factors including the pluripotent b-haemolysin/cytolysin (b-H/C). As GBS has the disposition of both a commensal organism and an invasive pathogen, it is important for the organism to appropriately regulate b-H/C and other virulence factors in response to the environment. GBS can repress transcription of b-H/C using the two-component system, CovR/CovS. Recently, we described that the serine/ threonine kinase Stk1 can phosphorylate CovR at threonine 65 to relieve repression of b-H/C. In this study, we show that infection with CovR-deficient GBS strains resulted in increased sepsis. Although CovRdeficient GBS showed decreased ability to invade the brain endothelium in vitro, they were more proficient in induction of permeability and pro-inflammatory signalling pathways in brain endothelium and penetration of the blood-brain barrier (BBB) in vivo. Microarray analysis revealed that CovR positively regulates its own expression and regulates the expression of 153 genes. Collectively, our results suggest that the positive feedback loop which regulates CovR transcription modulates host cell interaction and immune defence and may facilitate the transition of GBS from a commensal organism to a virulent meningeal pathogen.

Clinical research in infectious diseases

Group B Streptococci (GBS) are ß-hemolytic, gram-positive bacteria that are typically associated ... more Group B Streptococci (GBS) are ß-hemolytic, gram-positive bacteria that are typically associated with infections in human newborns or immunocompromised adults. However, mutation in the two-component regulator CovR/S relieves repression of hemolysin, potentially increasing virulence of GBS. We report the isolation of hyperhemolytic/hyperpigmented GBS strain from an adolescent patient who presented to the University of Washington clinic with symptoms of sore throat. While the patient also tested positive for mononucleosis, a GBS strain with increased hemolysis was isolated from the throat swab obtained from the patient. As hyperhemolytic/hyperpigmented GBS strains are typically associated with mutations in the regulator CovR/CovS, we sequenced the covR/S loci in the clinical isolate. An adenine to cytosine mutation resulting in a change in amino acid coding sequence from glutamine at position 120 to proline in CovR (Q120P) was identified. Introduction of the Q120P amino acid substitut...

Pathogens (Basel, Switzerland), Jan 22, 2015

Staphylococcus aureus are Gram-positive bacteria that are the leading cause of recurrent infectio... more Staphylococcus aureus are Gram-positive bacteria that are the leading cause of recurrent infections in humans that include pneumonia, bacteremia, osteomyelitis, arthritis, endocarditis, and toxic shock syndrome. The emergence of methicillin resistant S. aureus strains (MRSA) has imposed a significant concern in sustained measures of treatment against these infections. Recently, MRSA strains deficient in expression of a serine/threonine kinase (Stk1 or PknB) were described to exhibit increased sensitivity to β-lactam antibiotics. In this study, we screened a library consisting of 280 drug-like, low-molecular-weight compounds with the ability to inhibit protein kinases for those that increased the sensitivity of wild-type MRSA to β-lactams and then evaluated their toxicity in mice. We report the identification of four kinase inhibitors, the sulfonamides ST085384, ST085404, ST085405, and ST085399 that increased sensitivity of WT MRSA to sub-lethal concentrations of β-lactams. Furthermo...

Science Advances, 2015

Ascending infection of microbes from the lower genital tract into the amniotic cavity increases t... more Ascending infection of microbes from the lower genital tract into the amniotic cavity increases the risk of preterm birth, stillbirth, and newborn infections. Host defenses that are critical for preventing ascending microbial infection are not completely understood. Group B Streptococcus (GBS) are Gram-positive bacteria that frequently colonize the lower genital tract of healthy women but cause severe infections during pregnancy, leading to preterm birth, stillbirth, or early-onset newborn infections. We recently described that the GBS pigment is hemolytic, and increased pigment expression promotes GBS penetration of human placenta. Here, we show that the GBS hemolytic pigment/lipid toxin and hyperpigmented GBS strains induce mast cell degranulation, leading to the release of preformed and proinflammatory mediators. Mast cell-deficient mice exhibit enhanced bacterial burden, decreased neutrophil mobilization, and decreased immune responses during systemic GBS infection. In a vaginal colonization model, hyperpigmented GBS strains showed increased persistence in mast cell-deficient mice compared to mast cell-proficient mice. Consistent with these observations, fewer rectovaginal GBS isolates from women in their third trimester of pregnancy were hyperpigmented/hyperhemolytic. Our work represents the first example of a bacterial hemolytic lipid that induces mast cell degranulation and emphasizes the role of mast cells in limiting genital colonization by hyperpigmented GBS.

Infection and Immunity, 2015

Group B streptococci (GBS; Streptococcus agalactiae) are beta-hemolytic, Gram-positive bacteria t... more Group B streptococci (GBS; Streptococcus agalactiae) are beta-hemolytic, Gram-positive bacteria that are common asymptomatic colonizers of healthy adults. However, these opportunistic bacteria also cause invasive infections in human newborns and in certain adult populations. To adapt to the various environments encountered during its disease cycle, GBS encodes a number of two-component signaling systems. Previous studies have indicated that the TCS comprising the sensor histidine kinase RgfC and the response regulator RgfA mediate GBS binding to extracellular matrix components, such as fibrinogen. However, in certain GBS clinical isolates, a point mutation in rgfA results in premature truncation of the response regulator. The truncated RgfA protein lacks the C-terminal DNA binding domain necessary for promoter binding and gene regulation. Here, we show that deletion of rgfC in GBS strains lacking a functional RgfA increased systemic infection. Furthermore, infection with the rgfC mutant increased induction of proinflammatory signaling pathways in vivo. Phosphoproteomic analysis revealed that 19 phosphopeptides corresponding to 12 proteins were differentially phosphorylated at aspartate, cysteine, serine, threonine, or tyrosine residues in the rgfC mutant. This included aspartate phosphorylation of a tyrosine kinase, CpsD, and a transcriptional regulator. Consistent with this observation, microarray analysis of the rgfC mutant indicated that >200 genes showed altered expression compared to the isogenic wild-type strain and included transcriptional regulators, transporters, and genes previously associated with GBS pathogenesis. Our observations suggest that in the absence of RgfA, nonspecific RgfC signaling affects the expression of virulence factors and GBS pathogenesis. WA, Rajagopal L. 2015. The sensor histidine kinase RgfC affects group B streptococcal virulence factor expression independent of its response regulator RgfA.

PLoS ONE, 2010

Exotoxins, including the hemolysins known as the alpha (a) and beta (b) toxins, play an important... more Exotoxins, including the hemolysins known as the alpha (a) and beta (b) toxins, play an important role in the pathogenesis of Staphylococcus aureus infections. A random transposon library was screened for S. aureus mutants exhibiting altered hemolysin expression compared to wild type. Transposon insertions in 72 genes resulting in increased or decreased hemolysin expression were identified. Mutations inactivating a putative cyclic di-GMP synthetase and a serine/threonine phosphatase (Stp1) were found to reduce hemolysin expression, and mutations in genes encoding a two component regulator PhoR, LysR family transcriptional regulator, purine biosynthetic enzymes and a serine/threonine kinase (Stk1) increased expression. Transcription of the hla gene encoding a toxin was decreased in a Dstp1 mutant strain and increased in a Dstk1 strain. Microarray analysis of a Dstk1 mutant revealed increased transcription of additional exotoxins. A Dstp1 strain is severely attenuated for virulence in mice and elicits less inflammation and IL-6 production than the Dstk1 strain. In vivo phosphopeptide enrichment and mass spectrometric analysis revealed that threonine phosphorylated peptides corresponding to Stk1, DNA binding histone like protein (HU), serine-aspartate rich fibrinogen/bone sialoprotein binding protein (SdrE) and a hypothetical protein (NWMN_1123) were present in the wild type and not in the Dstk1 mutant. Collectively, these studies suggest that Stk1 mediated phosphorylation of HU, SrdE and NWMN_1123 affects S. aureus gene expression and virulence. Citation: Burnside K, Lembo A, de los Reyes M, Iliuk A, BinhTran N-T, et al. (2010) Regulation of Hemolysin Expression and Virulence of Staphylococcus aureus by a Serine/Threonine Kinase and Phosphatase. PLoS ONE 5(6): e11071.

Virology, 2006

Retroperitoneal fibromatosis herpesvirus (RFHV), the macaque homolog of the human rhadinovirus, K... more Retroperitoneal fibromatosis herpesvirus (RFHV), the macaque homolog of the human rhadinovirus, Kaposi's sarcoma-associated herpesvirus (KSHV), was first identified in retroperitoneal fibromatosis (RF) tumor lesions of macaques with simian AIDS. We cloned and sequenced the ORF73 latency-associated nuclear antigen (LANA) of RFHVMn from the pig-tailed macaque. RFHVMn LANA is structurally analogous to KSHV ORF73 LANA and contains an N-terminal serine-proline-rich region, a large internal glutamic acidic-rich repeat region and a conserved C-terminal domain. RFHVMn LANA reacts with monoclonal antibodies specific for a glutamic acid-proline dipeptide motif and a glutamic acidglutamine-rich motif in the KSHV LANA repeat region. Immunohistochemical and immunofluorescence analysis revealed that RFHVMn LANA is a nuclear antigen which is highly expressed in RF spindloid tumor cells. These data suggest that RFHV LANA is an ortholog of KSHV LANA and will function similarly to maintain viral latency and play a role in tumorigenicity in macaques.

PLoS ONE, 2011

ORF73 latency-associated nuclear antigen (LANA) of the Kaposi's sarcoma-associated herpesvirus (K... more ORF73 latency-associated nuclear antigen (LANA) of the Kaposi's sarcoma-associated herpesvirus (KSHV) is targeted to the nucleus of infected cells where it binds to chromatin and mediates viral episome persistence, interacts with cellular proteins and plays a role in latency and tumorigenesis. A structurally related LANA homolog has been identified in the retroperitoneal fibromatosis herpesvirus (RFHV), the macaque homolog of KSHV. Here, we report the evolutionary and functional conservation of a novel bi-functional nuclear localization signal (NLS) in KSHV and RFHV LANA. N-terminal peptides from both proteins were fused to EGFP or double EGFP fusions to examine their ability to induce nuclear transport of a heterologous protein. In addition, GST-pull down experiments were used to analyze the ability of LANA peptides to interact with members of the karyopherin family of nuclear transport receptors. Our studies revealed that both LANA proteins contain an N-terminal arginine/glycine (RG)-rich domain spanning a conserved chromatin-binding motif, which binds directly to importin b1 in a RanGTP-sensitive manner and serves as an NLS in the importin b1-mediated non-classical nuclear import pathway. Embedded within this domain is a conserved lysine/arginine-(KR)-rich bipartite motif that binds directly to multiple members of the importin a family of nuclear import adaptors in a RanGTP-insensitive manner and serves as an NLS in the classical importin a/b-mediated nuclear import pathway. The positioning of a classical bipartite kr-NLS embedded within a non-classical rg-NLS is a unique arrangement in these viral proteins, whose nuclear localization is critical to their functionality and to the virus life cycle. The ability to interact with multiple import receptors provides alternate pathways for nuclear localization of LANA. Since different import receptors can import cargo to distinct subnuclear compartments, a multifunctional NLS may provide LANA with an increased ability to interact with different nuclear components in its multifunctional role to maintain viral latency.

Molecular Microbiology, 2009

Molecular Microbiology, 2010

The pathogen encodes a number of virulence factors including the pluripotent b-haemolysin/cytolys... more The pathogen encodes a number of virulence factors including the pluripotent b-haemolysin/cytolysin (b-H/C). As GBS has the disposition of both a commensal organism and an invasive pathogen, it is important for the organism to appropriately regulate b-H/C and other virulence factors in response to the environment. GBS can repress transcription of b-H/C using the two-component system, CovR/CovS. Recently, we described that the serine/ threonine kinase Stk1 can phosphorylate CovR at threonine 65 to relieve repression of b-H/C. In this study, we show that infection with CovR-deficient GBS strains resulted in increased sepsis. Although CovRdeficient GBS showed decreased ability to invade the brain endothelium in vitro, they were more proficient in induction of permeability and pro-inflammatory signalling pathways in brain endothelium and penetration of the blood-brain barrier (BBB) in vivo. Microarray analysis revealed that CovR positively regulates its own expression and regulates the expression of 153 genes. Collectively, our results suggest that the positive feedback loop which regulates CovR transcription modulates host cell interaction and immune defence and may facilitate the transition of GBS from a commensal organism to a virulent meningeal pathogen.

Journal of Infectious Diseases, 2012

Staphylococcus aureus are gram-positive bacteria that cause clinically significant infections in ... more Staphylococcus aureus are gram-positive bacteria that cause clinically significant infections in humans. Severe S. aureus infections are particularly problematic in hospitalized patients and reoccur despite therapeutic measures. The absence of natural protective immune responses and the lack of high-throughput approaches to identify S. aureus antigens have imposed constraints in the development of effective vaccines. Here, we showed that vaccination with the genetically attenuated S. aureus mutant, inactivated using UV irradiation rather than heat, significantly increased survival and diminished bacterial burden and kidney abscesses when mice were challenged with virulent methicillin-sensitive or methicillin-resistant S. aureus. Protection conferred by immunization could be transferred to the naive host and was not observed in B-cell-deficient mice. Using a novel S. aureus whole-proteome microarray, we show that immunoglobulin G antibody responses to 83 proteins were observed in the immunized mice. These results suggest that protection against S. aureus infections requires antibody responses to the wide repertoire of antigens/virulence factors. Vaccination using UV-irradiated genetically attenuated S. aureus induces humoral immunity and provides a vaccine strategy for pathogens that fail to induce protective immunity. We also describe a novel, high-throughput technology to easily identify S. aureus antigens for vaccine development.

Journal of Experimental Medicine, 2013

are hyperhemolytic and some are associated with covR/S mutations. We demonstrate for the first ti... more are hyperhemolytic and some are associated with covR/S mutations. We demonstrate for the first time that hemolytic and cytolytic activity of GBS is due to the ornithine rhamnolipid pigment and not due to a pore-forming protein toxin. Our studies emphasize the importance of the hemolytic GBS pigment in ascending infection and fetal injury.

Journal of Biological Chemistry, 2011

Signaling mediated by serine/threonine phosphatases during bacterial pathogenesis is not complete... more Signaling mediated by serine/threonine phosphatases during bacterial pathogenesis is not completely understood. Results: In Group B Streptococcus (GBS), Stp1 controls serine/threonine kinase function, posttranscriptional regulation of hemolysin, autolysis and virulence. Conclusion: Although not essential for growth, Stp1 is critical for GBS pathogenesis. Significance: The importance of Stp1 in virulence and autolysis accentuates the possibility of using phosphatase inhibitors to decrease GBS infections.

Journal of Bacteriology, 2003

The cell wall of the environmental pathogen Mycobacterium avium is important to its virulence and... more The cell wall of the environmental pathogen Mycobacterium avium is important to its virulence and intrinsic antimicrobial resistance. To identify genes involved in cell wall biosynthesis, "transposome" insertion libraries were screened for mutants with altered colony morphology on medium containing the lipoprotein stain Congo red. Nineteen such mutants were isolated and mapped, including 10 with insertions in a functional island of cell wall biosynthetic genes that spans approximately 40 kb of the M. avium genome.

Future Microbiology, 2011

Living organisms adapt to the dynamic external environment for their survival. Environmental adap... more Living organisms adapt to the dynamic external environment for their survival. Environmental adaptation in prokaryotes is thought to be primarily accomplished by signaling events mediated by two-component systems, consisting of histidine kinases and response regulators. However, eukaryotic-like serine/threonine kinases (STKs) have recently been described to regulate growth, antibiotic resistance and virulence of pathogenic bacteria. This article summarizes the role of STKs and their cognate phosphatases (STPs) in Gram-positive cocci that cause invasive infections in humans. Given that a large number of inhibitors to eukaryotic STKs are approved for use in humans, understanding how serine/threonine phosphorylation regulates virulence and antibiotic resistance will be beneficial for the development of novel therapeutic strategies against bacterial infections.

Current Opinion in Microbiology, 2012

A growing body of evidence indicates that serine/threonine kinases (STKs) and phosphatases (STPs)... more A growing body of evidence indicates that serine/threonine kinases (STKs) and phosphatases (STPs) regulate gene expression in prokaryotic organisms. As prokaryotic STKs and STPs are not DNA binding proteins, regulation of gene expression is accomplished through post-translational modification of their targets. These include two-component response regulators, DNA binding proteins and proteins that mediate transcription and translation. This review summarizes our current understanding of how STKs and STPs mediate gene expression in prokaryotes. Further studies to identify environmental signals that trigger the signaling cascade and elucidation of mechanisms that regulate crosstalk between eukaryotic-like signaling enzymes, two-component systems, and components of the transcriptional and translational machinery will facilitate a greater understanding of prokaryotic gene regulation.

Virology Journal, 2009

Background: ORF59 DNA polymerase processivity factor of the human rhadinovirus, Kaposi's sarcoma-... more Background: ORF59 DNA polymerase processivity factor of the human rhadinovirus, Kaposi's sarcoma-associated herpesvirus (KSHV), is required for efficient copying of the genome during virus replication. KSHV ORF59 is antigenic in the infected host and is used as a marker for virus activation and replication.

Journal of Bacteriology, 2003

Molecular Microbiology, May 1, 2010

The pathogen encodes a number of virulence factors including the pluripotent b-haemolysin/cytolys... more The pathogen encodes a number of virulence factors including the pluripotent b-haemolysin/cytolysin (b-H/C). As GBS has the disposition of both a commensal organism and an invasive pathogen, it is important for the organism to appropriately regulate b-H/C and other virulence factors in response to the environment. GBS can repress transcription of b-H/C using the two-component system, CovR/CovS. Recently, we described that the serine/ threonine kinase Stk1 can phosphorylate CovR at threonine 65 to relieve repression of b-H/C. In this study, we show that infection with CovR-deficient GBS strains resulted in increased sepsis. Although CovRdeficient GBS showed decreased ability to invade the brain endothelium in vitro, they were more proficient in induction of permeability and pro-inflammatory signalling pathways in brain endothelium and penetration of the blood-brain barrier (BBB) in vivo. Microarray analysis revealed that CovR positively regulates its own expression and regulates the expression of 153 genes. Collectively, our results suggest that the positive feedback loop which regulates CovR transcription modulates host cell interaction and immune defence and may facilitate the transition of GBS from a commensal organism to a virulent meningeal pathogen.

Clinical research in infectious diseases

Group B Streptococci (GBS) are ß-hemolytic, gram-positive bacteria that are typically associated ... more Group B Streptococci (GBS) are ß-hemolytic, gram-positive bacteria that are typically associated with infections in human newborns or immunocompromised adults. However, mutation in the two-component regulator CovR/S relieves repression of hemolysin, potentially increasing virulence of GBS. We report the isolation of hyperhemolytic/hyperpigmented GBS strain from an adolescent patient who presented to the University of Washington clinic with symptoms of sore throat. While the patient also tested positive for mononucleosis, a GBS strain with increased hemolysis was isolated from the throat swab obtained from the patient. As hyperhemolytic/hyperpigmented GBS strains are typically associated with mutations in the regulator CovR/CovS, we sequenced the covR/S loci in the clinical isolate. An adenine to cytosine mutation resulting in a change in amino acid coding sequence from glutamine at position 120 to proline in CovR (Q120P) was identified. Introduction of the Q120P amino acid substitut...

Pathogens (Basel, Switzerland), Jan 22, 2015

Staphylococcus aureus are Gram-positive bacteria that are the leading cause of recurrent infectio... more Staphylococcus aureus are Gram-positive bacteria that are the leading cause of recurrent infections in humans that include pneumonia, bacteremia, osteomyelitis, arthritis, endocarditis, and toxic shock syndrome. The emergence of methicillin resistant S. aureus strains (MRSA) has imposed a significant concern in sustained measures of treatment against these infections. Recently, MRSA strains deficient in expression of a serine/threonine kinase (Stk1 or PknB) were described to exhibit increased sensitivity to β-lactam antibiotics. In this study, we screened a library consisting of 280 drug-like, low-molecular-weight compounds with the ability to inhibit protein kinases for those that increased the sensitivity of wild-type MRSA to β-lactams and then evaluated their toxicity in mice. We report the identification of four kinase inhibitors, the sulfonamides ST085384, ST085404, ST085405, and ST085399 that increased sensitivity of WT MRSA to sub-lethal concentrations of β-lactams. Furthermo...

Science Advances, 2015

Ascending infection of microbes from the lower genital tract into the amniotic cavity increases t... more Ascending infection of microbes from the lower genital tract into the amniotic cavity increases the risk of preterm birth, stillbirth, and newborn infections. Host defenses that are critical for preventing ascending microbial infection are not completely understood. Group B Streptococcus (GBS) are Gram-positive bacteria that frequently colonize the lower genital tract of healthy women but cause severe infections during pregnancy, leading to preterm birth, stillbirth, or early-onset newborn infections. We recently described that the GBS pigment is hemolytic, and increased pigment expression promotes GBS penetration of human placenta. Here, we show that the GBS hemolytic pigment/lipid toxin and hyperpigmented GBS strains induce mast cell degranulation, leading to the release of preformed and proinflammatory mediators. Mast cell-deficient mice exhibit enhanced bacterial burden, decreased neutrophil mobilization, and decreased immune responses during systemic GBS infection. In a vaginal colonization model, hyperpigmented GBS strains showed increased persistence in mast cell-deficient mice compared to mast cell-proficient mice. Consistent with these observations, fewer rectovaginal GBS isolates from women in their third trimester of pregnancy were hyperpigmented/hyperhemolytic. Our work represents the first example of a bacterial hemolytic lipid that induces mast cell degranulation and emphasizes the role of mast cells in limiting genital colonization by hyperpigmented GBS.

Infection and Immunity, 2015

Group B streptococci (GBS; Streptococcus agalactiae) are beta-hemolytic, Gram-positive bacteria t... more Group B streptococci (GBS; Streptococcus agalactiae) are beta-hemolytic, Gram-positive bacteria that are common asymptomatic colonizers of healthy adults. However, these opportunistic bacteria also cause invasive infections in human newborns and in certain adult populations. To adapt to the various environments encountered during its disease cycle, GBS encodes a number of two-component signaling systems. Previous studies have indicated that the TCS comprising the sensor histidine kinase RgfC and the response regulator RgfA mediate GBS binding to extracellular matrix components, such as fibrinogen. However, in certain GBS clinical isolates, a point mutation in rgfA results in premature truncation of the response regulator. The truncated RgfA protein lacks the C-terminal DNA binding domain necessary for promoter binding and gene regulation. Here, we show that deletion of rgfC in GBS strains lacking a functional RgfA increased systemic infection. Furthermore, infection with the rgfC mutant increased induction of proinflammatory signaling pathways in vivo. Phosphoproteomic analysis revealed that 19 phosphopeptides corresponding to 12 proteins were differentially phosphorylated at aspartate, cysteine, serine, threonine, or tyrosine residues in the rgfC mutant. This included aspartate phosphorylation of a tyrosine kinase, CpsD, and a transcriptional regulator. Consistent with this observation, microarray analysis of the rgfC mutant indicated that >200 genes showed altered expression compared to the isogenic wild-type strain and included transcriptional regulators, transporters, and genes previously associated with GBS pathogenesis. Our observations suggest that in the absence of RgfA, nonspecific RgfC signaling affects the expression of virulence factors and GBS pathogenesis. WA, Rajagopal L. 2015. The sensor histidine kinase RgfC affects group B streptococcal virulence factor expression independent of its response regulator RgfA.

PLoS ONE, 2010

Exotoxins, including the hemolysins known as the alpha (a) and beta (b) toxins, play an important... more Exotoxins, including the hemolysins known as the alpha (a) and beta (b) toxins, play an important role in the pathogenesis of Staphylococcus aureus infections. A random transposon library was screened for S. aureus mutants exhibiting altered hemolysin expression compared to wild type. Transposon insertions in 72 genes resulting in increased or decreased hemolysin expression were identified. Mutations inactivating a putative cyclic di-GMP synthetase and a serine/threonine phosphatase (Stp1) were found to reduce hemolysin expression, and mutations in genes encoding a two component regulator PhoR, LysR family transcriptional regulator, purine biosynthetic enzymes and a serine/threonine kinase (Stk1) increased expression. Transcription of the hla gene encoding a toxin was decreased in a Dstp1 mutant strain and increased in a Dstk1 strain. Microarray analysis of a Dstk1 mutant revealed increased transcription of additional exotoxins. A Dstp1 strain is severely attenuated for virulence in mice and elicits less inflammation and IL-6 production than the Dstk1 strain. In vivo phosphopeptide enrichment and mass spectrometric analysis revealed that threonine phosphorylated peptides corresponding to Stk1, DNA binding histone like protein (HU), serine-aspartate rich fibrinogen/bone sialoprotein binding protein (SdrE) and a hypothetical protein (NWMN_1123) were present in the wild type and not in the Dstk1 mutant. Collectively, these studies suggest that Stk1 mediated phosphorylation of HU, SrdE and NWMN_1123 affects S. aureus gene expression and virulence. Citation: Burnside K, Lembo A, de los Reyes M, Iliuk A, BinhTran N-T, et al. (2010) Regulation of Hemolysin Expression and Virulence of Staphylococcus aureus by a Serine/Threonine Kinase and Phosphatase. PLoS ONE 5(6): e11071.

Virology, 2006

Retroperitoneal fibromatosis herpesvirus (RFHV), the macaque homolog of the human rhadinovirus, K... more Retroperitoneal fibromatosis herpesvirus (RFHV), the macaque homolog of the human rhadinovirus, Kaposi's sarcoma-associated herpesvirus (KSHV), was first identified in retroperitoneal fibromatosis (RF) tumor lesions of macaques with simian AIDS. We cloned and sequenced the ORF73 latency-associated nuclear antigen (LANA) of RFHVMn from the pig-tailed macaque. RFHVMn LANA is structurally analogous to KSHV ORF73 LANA and contains an N-terminal serine-proline-rich region, a large internal glutamic acidic-rich repeat region and a conserved C-terminal domain. RFHVMn LANA reacts with monoclonal antibodies specific for a glutamic acid-proline dipeptide motif and a glutamic acidglutamine-rich motif in the KSHV LANA repeat region. Immunohistochemical and immunofluorescence analysis revealed that RFHVMn LANA is a nuclear antigen which is highly expressed in RF spindloid tumor cells. These data suggest that RFHV LANA is an ortholog of KSHV LANA and will function similarly to maintain viral latency and play a role in tumorigenicity in macaques.

PLoS ONE, 2011

ORF73 latency-associated nuclear antigen (LANA) of the Kaposi's sarcoma-associated herpesvirus (K... more ORF73 latency-associated nuclear antigen (LANA) of the Kaposi's sarcoma-associated herpesvirus (KSHV) is targeted to the nucleus of infected cells where it binds to chromatin and mediates viral episome persistence, interacts with cellular proteins and plays a role in latency and tumorigenesis. A structurally related LANA homolog has been identified in the retroperitoneal fibromatosis herpesvirus (RFHV), the macaque homolog of KSHV. Here, we report the evolutionary and functional conservation of a novel bi-functional nuclear localization signal (NLS) in KSHV and RFHV LANA. N-terminal peptides from both proteins were fused to EGFP or double EGFP fusions to examine their ability to induce nuclear transport of a heterologous protein. In addition, GST-pull down experiments were used to analyze the ability of LANA peptides to interact with members of the karyopherin family of nuclear transport receptors. Our studies revealed that both LANA proteins contain an N-terminal arginine/glycine (RG)-rich domain spanning a conserved chromatin-binding motif, which binds directly to importin b1 in a RanGTP-sensitive manner and serves as an NLS in the importin b1-mediated non-classical nuclear import pathway. Embedded within this domain is a conserved lysine/arginine-(KR)-rich bipartite motif that binds directly to multiple members of the importin a family of nuclear import adaptors in a RanGTP-insensitive manner and serves as an NLS in the classical importin a/b-mediated nuclear import pathway. The positioning of a classical bipartite kr-NLS embedded within a non-classical rg-NLS is a unique arrangement in these viral proteins, whose nuclear localization is critical to their functionality and to the virus life cycle. The ability to interact with multiple import receptors provides alternate pathways for nuclear localization of LANA. Since different import receptors can import cargo to distinct subnuclear compartments, a multifunctional NLS may provide LANA with an increased ability to interact with different nuclear components in its multifunctional role to maintain viral latency.

Molecular Microbiology, 2009

Molecular Microbiology, 2010

The pathogen encodes a number of virulence factors including the pluripotent b-haemolysin/cytolys... more The pathogen encodes a number of virulence factors including the pluripotent b-haemolysin/cytolysin (b-H/C). As GBS has the disposition of both a commensal organism and an invasive pathogen, it is important for the organism to appropriately regulate b-H/C and other virulence factors in response to the environment. GBS can repress transcription of b-H/C using the two-component system, CovR/CovS. Recently, we described that the serine/ threonine kinase Stk1 can phosphorylate CovR at threonine 65 to relieve repression of b-H/C. In this study, we show that infection with CovR-deficient GBS strains resulted in increased sepsis. Although CovRdeficient GBS showed decreased ability to invade the brain endothelium in vitro, they were more proficient in induction of permeability and pro-inflammatory signalling pathways in brain endothelium and penetration of the blood-brain barrier (BBB) in vivo. Microarray analysis revealed that CovR positively regulates its own expression and regulates the expression of 153 genes. Collectively, our results suggest that the positive feedback loop which regulates CovR transcription modulates host cell interaction and immune defence and may facilitate the transition of GBS from a commensal organism to a virulent meningeal pathogen.

Journal of Infectious Diseases, 2012

Staphylococcus aureus are gram-positive bacteria that cause clinically significant infections in ... more Staphylococcus aureus are gram-positive bacteria that cause clinically significant infections in humans. Severe S. aureus infections are particularly problematic in hospitalized patients and reoccur despite therapeutic measures. The absence of natural protective immune responses and the lack of high-throughput approaches to identify S. aureus antigens have imposed constraints in the development of effective vaccines. Here, we showed that vaccination with the genetically attenuated S. aureus mutant, inactivated using UV irradiation rather than heat, significantly increased survival and diminished bacterial burden and kidney abscesses when mice were challenged with virulent methicillin-sensitive or methicillin-resistant S. aureus. Protection conferred by immunization could be transferred to the naive host and was not observed in B-cell-deficient mice. Using a novel S. aureus whole-proteome microarray, we show that immunoglobulin G antibody responses to 83 proteins were observed in the immunized mice. These results suggest that protection against S. aureus infections requires antibody responses to the wide repertoire of antigens/virulence factors. Vaccination using UV-irradiated genetically attenuated S. aureus induces humoral immunity and provides a vaccine strategy for pathogens that fail to induce protective immunity. We also describe a novel, high-throughput technology to easily identify S. aureus antigens for vaccine development.

Journal of Experimental Medicine, 2013

are hyperhemolytic and some are associated with covR/S mutations. We demonstrate for the first ti... more are hyperhemolytic and some are associated with covR/S mutations. We demonstrate for the first time that hemolytic and cytolytic activity of GBS is due to the ornithine rhamnolipid pigment and not due to a pore-forming protein toxin. Our studies emphasize the importance of the hemolytic GBS pigment in ascending infection and fetal injury.

Journal of Biological Chemistry, 2011

Signaling mediated by serine/threonine phosphatases during bacterial pathogenesis is not complete... more Signaling mediated by serine/threonine phosphatases during bacterial pathogenesis is not completely understood. Results: In Group B Streptococcus (GBS), Stp1 controls serine/threonine kinase function, posttranscriptional regulation of hemolysin, autolysis and virulence. Conclusion: Although not essential for growth, Stp1 is critical for GBS pathogenesis. Significance: The importance of Stp1 in virulence and autolysis accentuates the possibility of using phosphatase inhibitors to decrease GBS infections.

Journal of Bacteriology, 2003

The cell wall of the environmental pathogen Mycobacterium avium is important to its virulence and... more The cell wall of the environmental pathogen Mycobacterium avium is important to its virulence and intrinsic antimicrobial resistance. To identify genes involved in cell wall biosynthesis, "transposome" insertion libraries were screened for mutants with altered colony morphology on medium containing the lipoprotein stain Congo red. Nineteen such mutants were isolated and mapped, including 10 with insertions in a functional island of cell wall biosynthetic genes that spans approximately 40 kb of the M. avium genome.

Future Microbiology, 2011

Living organisms adapt to the dynamic external environment for their survival. Environmental adap... more Living organisms adapt to the dynamic external environment for their survival. Environmental adaptation in prokaryotes is thought to be primarily accomplished by signaling events mediated by two-component systems, consisting of histidine kinases and response regulators. However, eukaryotic-like serine/threonine kinases (STKs) have recently been described to regulate growth, antibiotic resistance and virulence of pathogenic bacteria. This article summarizes the role of STKs and their cognate phosphatases (STPs) in Gram-positive cocci that cause invasive infections in humans. Given that a large number of inhibitors to eukaryotic STKs are approved for use in humans, understanding how serine/threonine phosphorylation regulates virulence and antibiotic resistance will be beneficial for the development of novel therapeutic strategies against bacterial infections.

Current Opinion in Microbiology, 2012

A growing body of evidence indicates that serine/threonine kinases (STKs) and phosphatases (STPs)... more A growing body of evidence indicates that serine/threonine kinases (STKs) and phosphatases (STPs) regulate gene expression in prokaryotic organisms. As prokaryotic STKs and STPs are not DNA binding proteins, regulation of gene expression is accomplished through post-translational modification of their targets. These include two-component response regulators, DNA binding proteins and proteins that mediate transcription and translation. This review summarizes our current understanding of how STKs and STPs mediate gene expression in prokaryotes. Further studies to identify environmental signals that trigger the signaling cascade and elucidation of mechanisms that regulate crosstalk between eukaryotic-like signaling enzymes, two-component systems, and components of the transcriptional and translational machinery will facilitate a greater understanding of prokaryotic gene regulation.

Virology Journal, 2009

Background: ORF59 DNA polymerase processivity factor of the human rhadinovirus, Kaposi's sarcoma-... more Background: ORF59 DNA polymerase processivity factor of the human rhadinovirus, Kaposi's sarcoma-associated herpesvirus (KSHV), is required for efficient copying of the genome during virus replication. KSHV ORF59 is antigenic in the infected host and is used as a marker for virus activation and replication.