Exploring the Pregnant Guinea Pig as a Model for Group B Streptococcus Intrauterine Infection.

Infection of the amniotic cavity remains a major cause of preterm birth, stillbirth, fetal injury and early onset, fulminant infections in newborns. Currently, there are no effective therapies to prevent in utero infection and consequent co-morbidities. This is in part due to the lack of feasible and appropriate animal models to understand mechanisms that lead to in utero infections. Use of mouse and rat models do not fully recapitulate human pregnancy, while pregnant nonhuman primate models are limited by ethical considerations, technical constraints, and cost. Given these limitations, the guinea pig is an attractive animal model for studying pregnancy infections, particularly as the placental structure is quite similar to the human placenta. Here, we describe our studies that explored the pregnant guinea pig as a model to study in utero Group B Streptococci (GBS) infections. We observed that intrauterine inoculation of wild type GBS in pregnant guinea pigs resulted in bacterial invasion and dissemination to the placenta, amniotic fluid and fetal organs. Also, hyperhemolytic GBS such as those lacking the hemolysin repressor CovR/S showed increased dissemination into the amniotic fluid and fetal organs such as the fetal lung and brain. These results are similar to those observed in mouse and non-human primate models of in utero infection, and support use of the guinea pig as a model for studying GBS infections in pregnancy.

Kinase Inhibitors that Increase the Sensitivity of Methicillin Resistant Staphylococcus aureus to ß-Lactam Antibiotics.

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 ß-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 ß-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 ß-lactams. Furthermore, these inhibitors lacked alerting structures commonly associated with toxic effects, and toxicity was not observed with ST085384 or ST085405 in vivo in a murine model. These results suggest that kinase inhibitors may be useful in therapeutic strategies against MRSA infections.

Mast cell degranulation by a hemolytic lipid toxin decreases GBS colonization and infection.

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.

The sensor histidine kinase RgfC affects group B streptococcal virulence factor expression independent of its response regulator RgfA.

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.

A Hyperhemolytic/Hyperpigmented Group B Streptococcus Strain with a CovR Mutation Isolated from an Adolescent Patient with Sore Throat.

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 substitution in a CovR complementation plasmid abolished complementation of a ΔcovR mutant derived from the wild type GBS serotype Ia strain A909; these results confirm that the hyperhemolysis observed in the clinical isolate is due to the Q120P substitution in CovR. Antibiotic was prescribed and the patient's symptoms resolved without reported complications. This study represents the first report of the isolation of a hyperhemolytic/hyperpigmented GBS strain due to a covR/S mutation from an adolescent patient with persistent sore throat who was also diagnosed with mononucleosis. The isolation of GBS CovR/S mutants indicates their presence in settings of co-infections and includes adolescents.

A hemolytic pigment of Group B Streptococcus allows bacterial penetration of human placenta.

Microbial infection of the amniotic fluid is a significant cause of fetal injury, preterm birth, and newborn infections. Group B Streptococcus (GBS) is an important human bacterial pathogen associated with preterm birth, fetal injury, and neonatal mortality. Although GBS has been isolated from amniotic fluid of women in preterm labor, mechanisms of in utero infection remain unknown. Previous studies indicated that GBS are unable to invade human amniotic epithelial cells (hAECs), which represent the last barrier to the amniotic cavity and fetus. We show that GBS invades hAECs and strains lacking the hemolysin repressor CovR/S accelerate amniotic barrier failure and penetrate chorioamniotic membranes in a hemolysin-dependent manner. Clinical GBS isolates obtained from women in preterm labor 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.

Vaccination with a UV-irradiated genetically attenuated mutant of Staphylococcus aureus provides protection against subsequent systemic infection.

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.

Regulation of prokaryotic gene expression by eukaryotic-like enzymes.

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.

Serine/threonine phosphatase Stp1 mediates post-transcriptional regulation of hemolysin, autolysis, and virulence of group B Streptococcus.

Elucidating how serine/threonine phosphatases regulate kinase function and bacterial virulence is critical for our ability to combat these infections. Group B streptococci (GBS) are ß-hemolytic Gram-positive bacteria that cause invasive infections in humans. To adapt to environmental changes, GBS encodes signaling mechanisms comprising two component systems and eukaryotic-like enzymes. We have previously described the importance of the serine/threonine kinase Stk1 to GBS pathogenesis. However, how the presence or absence of the cognate serine/threonine phosphatase Stp1 affects Stk1 function and GBS virulence is not known. Here, we show that GBS deficient only in Stp1 expression are markedly reduced for their ability to cause systemic infections, exhibit decreased ß-hemolysin/cytolysin activity, and show increased sensitivity to autolysis. Although transcription of genes important for ß-hemolysin/cytolysin expression and export is similar to the wild type (WT), 294 genes (excluding stp1) showed altered expression in the stp1 mutant and included autolysin genes. Furthermore, phosphopeptide enrichment analysis identified that 35 serine/threonine phosphopeptides, corresponding to 27 proteins, were unique to the stp1 mutant. This included phosphorylation of ATP synthase, DNA and RNA helicases, and proteins important for cell division and protein synthesis. Collectively, our results indicate that Stp1 is important for appropriate regulation of Stk1 function, hemolysin activity, autolysis, and GBS virulence.

Aspects of eukaryotic-like signaling in Gram-positive cocci: a focus on virulence.

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.

Conservation of complex nuclear localization signals utilizing classical and non-classical nuclear import pathways in LANA homologs of KSHV and RFHV.

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 ß1 in a RanGTP-sensitive manner and serves as an NLS in the importin ß1-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 α family of nuclear import adaptors in a RanGTP-insensitive manner and serves as an NLS in the classical importin α/ß-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.

Regulation of hemolysin expression and virulence of Staphylococcus aureus by a serine/threonine kinase and phosphatase.

Exotoxins, including the hemolysins known as the alpha (alpha) and beta (beta) 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 alpha toxin was decreased in a Deltastp1 mutant strain and increased in a Deltastk1 strain. Microarray analysis of a Deltastk1 mutant revealed increased transcription of additional exotoxins. A Deltastp1 strain is severely attenuated for virulence in mice and elicits less inflammation and IL-6 production than the Deltastk1 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 Deltastk1 mutant. Collectively, these studies suggest that Stk1 mediated phosphorylation of HU, SrdE and NWMN_1123 affects S. aureus gene expression and virulence.

Regulation of CovR expression in Group B Streptococcus impacts blood-brain barrier penetration.

Group B Streptococcus (GBS) is an important cause of invasive infections in humans. The pathogen encodes a number of virulence factors including the pluripotent beta-haemolysin/cytolysin (beta-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 beta-H/C and other virulence factors in response to the environment. GBS can repress transcription of beta-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 beta-H/C. In this study, we show that infection with CovR-deficient GBS strains resulted in increased sepsis. Although CovR-deficient 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.

The ORF59 DNA polymerase processivity factor homologs of Old World primate RV2 rhadinoviruses are highly conserved nuclear antigens expressed in differentiated epithelium in infected macaques.

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. RESULTS: We cloned, sequenced and expressed the genes encoding related ORF59 proteins from the RV1 rhadinovirus homologs of KSHV from chimpanzee (PtrRV1) and three species of macaques (RFHVMm, RFHVMn and RFHVMf), and have compared them with ORF59 proteins obtained from members of the more distantly-related RV2 rhadinovirus lineage infecting the same non-human primate species (PtrRV2, RRV, MneRV2, and MfaRV2, respectively). We found that ORF59 homologs of the RV1 and RV2 Old World primate rhadinoviruses are highly conserved with distinct phylogenetic clustering of the two rhadinovirus lineages. RV1 and RV2 ORF59 C-terminal domains exhibit a strong lineage-specific conservation. Rabbit antiserum was developed against a C-terminal polypeptide that is highly conserved between the macaque RV2 ORF59 sequences. This anti-serum showed strong reactivity towards ORF59 encoded by the macaque RV2 rhadinoviruses, RRV (rhesus) and MneRV2 (pig-tail), with no cross reaction to human or macaque RV1 ORF59 proteins. Using this antiserum and RT-qPCR, we determined that RRV ORF59 is expressed early after permissive infection of both rhesus primary fetal fibroblasts and African green monkey kidney epithelial cells (Vero) in vitro. RRV- and MneRV2-infected foci showed strong nuclear expression of ORF59 that correlated with production of infectious progeny virus. Immunohistochemical studies of an MneRV2-infected macaque revealed strong nuclear expression of ORF59 in infected cells within the differentiating layer of epidermis corroborating previous observations that differentiated epithelial cells are permissive for replication of KSHV-like rhadinoviruses. CONCLUSION: The ORF59 DNA polymerase processivity factor homologs of the Old World primate RV1 and RV2 rhadinovirus lineages are phylogenetically distinct yet demonstrate similar expression and localization characteristics that correlate with their use as lineage-specific markers for permissive infection and virus replication. These studies will aid in the characterization of virus activation from latency to the replicative state, an important step for understanding the biology and transmission of rhadinoviruses, such as KSHV.

Threonine phosphorylation prevents promoter DNA binding of the Group B Streptococcus response regulator CovR.

All living organisms communicate with the external environment for their survival and existence. In prokaryotes, communication is achieved by two-component systems (TCS) comprising histidine kinases and response regulators. In eukaryotes, signalling is accomplished by serine/threonine and tyrosine kinases. Although TCS and serine/threonine kinases coexist in prokaryotes, direct cross-talk between these families was first described in Group B Streptococcus (GBS). A serine/threonine kinase (Stk1) and a TCS (CovR/CovS) co-regulate toxin expression in GBS. Typically, promoter binding of regulators like CovR is controlled by phosphorylation of the conserved active site aspartate (D53). In this study, we show that Stk1 phosphorylates CovR at threonine 65. The functional consequence of threonine phosphorylation of CovR in GBS was evaluated using phosphomimetic and silencing substitutions. GBS encoding the phosphomimetic T65E allele are deficient for CovR regulation unlike strains encoding the non-phosphorylated T65A allele. Further, compared with wild-type or T65A CovR, the T65E CovR is unable to bind promoter DNA and is decreased for phosphorylation at D53, similar to Stk1-phosphorylated CovR. Collectively, we provide evidence for a novel mechanism of response regulator control that enables GBS (and possibly other prokaryotes) to fine-tune gene expression for environmental adaptation.

RFHVMn ORF73 is structurally related to the KSHV ORF73 latency-associated nuclear antigen (LANA) and is expressed in retroperitoneal fibromatosis (RF) tumor cells.

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 acid-glutamine-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.

Mutational analysis of cell wall biosynthesis in Mycobacterium avium.

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.