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2.  Staphylococcus aureus Exploits Cathelicidin Antimicrobial Peptides Produced during Early Pneumonia to Promote Staphylokinase-Dependent Fibrinolysis 
The Journal of infectious diseases  2007;195(9):1365-1372.
The increasing prevalence of Staphylococcus aureus strains isolated from hospital- and community-acquired respiratory tract infections is an important public health concern worldwide. The majority of S. aureus strains produce staphylokinase, a plasminogen activator capable of inactivating neutrophil α-defensins and of impairing phagocytosis via opsonin degradation. Cathelicidin antimicrobial peptides are present at sites of infection before the release of neutrophil α-defensins. Therefore, we hypothesized that staphylokinase interacts with cathelicidin during the early pathogenesis of S. aureus airway infection. In a mouse intranasal infection model, cathelicidin was strongly up-regulated in the airways during the development of staphylococcal pneumonia. In vitro, cathelicidin bound directly to staphylokinase and augmented staphylokinase-dependent plasminogen activation and fibrinolysis at concentrations consistent with those detected in the airways during infection. These data suggest that staphylokinase production may be a novel virulence mechanism by which S. aureus exploits cathelicidin to promote fibrinolysis, leading to enhanced bacterial dissemination and invasive infection.
doi:10.1086/513277
PMCID: PMC2366818  PMID: 17397009
4.  Non-invasive whole genome sequencing of a human fetus 
Science Translational Medicine  2012;4(137):137ra76.
Analysis of cell-free fetal DNA in maternal plasma holds great promise for the development of non-invasive prenatal genetic diagnostics. However, previous studies have been restricted to detection of fetal trisomies (1, 2) or specific, paternally inherited mutations (3), or to genotyping common polymorphisms using invasively sampled material (4). Here, we combine genome sequencing of two parents, genome-wide maternal haplotyping (5), and deep sequencing of maternal plasma to non-invasively determine the genome sequence of a human fetus at 18.5 weeks gestation. Inheritance was predicted at 2.8×106 parentally heterozygous sites with 98.1% accuracy. Furthermore, 39 of 44 de novo point mutations in the fetal genome were detected, albeit with limited specificity. Subsampling these data and analyzing a second family trio by the same approach indicate that ~300 kilobase parental haplotype blocks combined with shallow sequencing of maternal plasma are sufficient to substantially determine the inherited complement of a fetal genome. However, ultra-deep sequencing of maternal plasma is necessary for the practical detection of fetal de novo mutations genome-wide. Although technical and analytical challenges remain, we anticipate that non-invasive analysis of inherited variation and de novo mutations in fetal genomes will facilitate the comprehensive prenatal diagnosis of both recessive and dominant Mendelian disorders.
doi:10.1126/scitranslmed.3004323
PMCID: PMC3379884  PMID: 22674554
5.  A novel streptococcal surface protease promotes virulence, resistance to opsonophagocytosis, and cleavage of human fibrinogen 
Group B streptococcus (GBS) is an important human pathogen. In this study, we sought to identify mechanisms that may protect GBS from host defenses in addition to its capsular polysaccharide. A gene encoding a cell-surface–associated protein (cspA) was characterized from a highly virulent type III GBS isolate, COH1. Its sequence indicated that it is a subtilisin-like extracellular serine protease homologous to streptococcal C5a peptidases and caseinases of lactic acid bacteria. The wild-type strain cleaved the α chain of human fibrinogen, whereas a cspA mutant, TOH121, was unable to cleave fibrinogen. We observed aggregated material when COH1 was incubated with fibrinogen but not when the mutant strain was treated similarly. This suggested that the product(s) of fibrinogen cleavage have strong adhesive properties and may be similar to fibrin. The cspA gene was present among representative clinical isolates from all nine capsular serotypes, as revealed by Southern blotting. A cspA– mutant was ten times less virulent in a neonatal rat sepsis model of GBS infections, as measured by LD50 analysis. In addition, the cspA– mutant was significantly more sensitive than the wild-type strain to opsonophagocytic killing by human neutrophils in vitro. Taken together, the results suggest that cleavage of fibrinogen by CspA may increase the lethality of GBS infection, potentially by protecting the bacterium from opsonophagocytic killing.
doi:10.1172/JCI200316270
PMCID: PMC151836  PMID: 12511589
6.  Serious and Life-Threatening Pregnancy-Related Infections: Opportunities to Reduce the Global Burden 
PLoS Medicine  2012;9(10):e1001324.
Michael Gravett and colleagues review the burden of pregnancy-related infections, especially in low- and middle-income countries, and offer suggestions for a more effective intervention strategy.
doi:10.1371/journal.pmed.1001324
PMCID: PMC3467240  PMID: 23055837
8.  Use of nonhuman primate models to investigate mechanisms of infection-associated preterm birth 
Preterm birth is the most important direct cause of neonatal mortality and remains a major challenge for obstetrics and global health. Intrauterine infection causes approximately 50% of early preterm births. Animal models using pregnant mice, rabbits, or sheep, demonstrate the key link between infection and premature birth, but differ in mechanisms of parturition and placental structure from humans. The nonhuman primate (NHP) is a powerful model which emulates many features of human placentation and parturition. The contributions of the NHP model to preterm birth research are reviewed emphasizing the role of infections, and potential development of preventative and therapeutic strategies.
doi:10.1111/j.1471-0528.2010.02728.x
PMCID: PMC3060081  PMID: 21040390
9.  Epidemiology and aetiology of maternal parasitic infections in low- and middle-income countries 
Journal of Global Health  2011;1(2):189-200.
Background
There have been very few systematic reviews looking at maternal infections in the developing world, even though cutting maternal mortality by three quarters is United Nation’s Millennium Development Goal number five. This systematic review has two aims. The first is to present the prevalence of parasitic infections in the developing world over the last 30 years and the second is to evaluate the quality and distribution of research in this field.
Methods
A systematic review of Medline, EMBASE and Global Health databases was undertaken using pre-determined search criteria. Three levels of quality criteria for exclusion of inadequate studies identified 115 out of initial 8580 titles. The data were extracted for 5 domains: worldwide pathogen prevalence, year of study, study setting, sample size and diagnostic test for each pathogen.
Results
The initial search retrieved 8580 results. From these titles, 43 studies on malaria, 12 studies on helminths, 49 studies on Toxoplasma gondii, 7 studies on Chagas disease, 5 studies on Trichomonas, 1 leishmaniasis study and 1 study on trichinellosis were extracted for analysis. High prevalence of malaria was found in Gabon (up to 57%) India (55%), Cameroon (50%), Yemen (55%), Nigeria (up to 64%) and Ghana (54%). High prevalence of hookworm infections was found in Nepal at 78.8% and high values of Ascaris lumbricoides were found in Nepal, (56.2%), Kenya (52.3%) and Gabon (45.5%). High levels of Schistosoma mansoni were found in Zimbabwe (50%) and Tanzania (63.5%). The prevalence of active Toxoplasma gondii infection was found to be highest in India (27.7%).
Conclusion
This study highlights the large burden of maternal parasitic infections globally. It may serve as a useful starting point for health policy development and research prioritization in this area.
PMCID: PMC3484768  PMID: 23198118
11.  Emerging biomarkers for the diagnosis of severe neonatal infections applicable to low resource settings 
Journal of Global Health  2011;1(2):210-223.
More than 500 000 children die each year in low resource settings due to serious neonatal infections. Better diagnostics that can be utilized in these settings to identify infected infants have the potential to significantly reduce neonatal deaths and the associated morbidity. A systematic review was performed and identified more than 250 potential new biomarkers for the diagnosis of serious neonatal infections. Eight of these biomarkers were both high-performance and high-abundance (antithrombin, inter-α inhibitor proteins, interferon-γ inducible protein-10, interleukin-1 receptor antagonist, LPS binding protein, mannose binding lectin, serum amyloid A, resistin, visfatin), and are promising for the diagnosis of serious neonatal infections in low resource settings. Future clinical trials comparing these biomarkers with more traditional biomarkers seem warranted.
PMCID: PMC3484779  PMID: 23198120
12.  Epidemiology and aetiology of maternal bacterial and viral infections in low- and middle-income countries 
Journal of Global Health  2011;1(2):171-188.
Background
Maternal morbidity and mortality in low- and middle-income countries has remained exceedingly high. However, information on bacterial and viral maternal infections, which are important contributors to poor pregnancy outcomes, is sparse and poorly characterised. This review aims to describe the epidemiology and aetiology of bacterial and viral maternal infections in low- and middle-income countries.
Methods
A systematic search of published literature was conducted and data on aetiology and epidemiology of maternal infections was extracted from relevant studies for analysis. Searches were conducted in parallel by two reviewers (using OVID) in the following databases: Medline (1950 to 2010), EMBASE (1980 to 2010) and Global Health (1973 to 2010).
Results
Data from 158 relevant studies was used to characterise the epidemiology of the 10 most extensively reported maternal infections with the following median prevalence rates: Treponema pallidum (2.6%), Neisseria gonorrhoeae (1.5%), Chlamydia trachomatis (5.8%), Group B Streptococcus (8.6%), bacterial vaginosis (20.9%), hepatitis B virus (4.3%), hepatitis C virus (1.4%), Cytomegalovirus (95.7% past infection), Rubella (8.9% susceptible) and Herpes simplex (20.7%). Large variations in the prevalence of these infections between countries and regions were noted.
Conclusion
This review confirms the suspected high prevalence of maternal bacterial and viral infections and identifies particular diseases and regions requiring urgent attention in public health policy planning, setting research priorities and donor funding towards reducing maternal morbidity and mortality in low- and middle-income countries.
PMCID: PMC3484781  PMID: 23198117
13.  Global report on preterm birth and stillbirth (4 of 7): delivery of interventions 
BMC Pregnancy and Childbirth  2010;10(Suppl 1):S4.
Background
The efficacious interventions identified in the previous article of this report will fail unless they are delivered at high and equitable coverage. This article discusses critical delivery constraints and strategies.
Barriers to scaling up interventions
Achieving universal coverage entails addressing major barriers at many levels. An overarching constraint is the lack of political will, resulting from the dearth of preterm birth and stillbirth data and the lack of visibility. Other barriers exist at the household and community levels, such as insufficient demand for interventions or sociocultural barriers; at the health services level, such as a lack of resources and trained healthcare providers; and at the health sector policy and management level, such as poorly functioning, centralized systems. Additional constraints involve weak governance and accountability, political instability, and challenges in the physical environment.
Strategies and examples
Scaling up maternal, newborn and child health interventions requires strengthening health systems, but there is also a role for focused, targeted interventions. Choosing a strategy involves identifying appropriate channels for reaching high coverage, which depends on many factors such as access to and attendance at healthcare facilities. Delivery channels vary, and may include facility- and community-based healthcare providers, mass media campaigns, and community-based approaches and marketing strategies. Issues related to scaling up are discussed in the context of four interventions that may be given to mothers at different stages throughout pregnancy or to newborns: (1) detection and treatment of syphilis; (2) emergency Cesarean section; (3) newborn resuscitation; and (4) kangaroo mother care. Systematic reviews of the literature and large-scale implementation studies are analyzed for each intervention.
Conclusion
Equitable and successful scale-up of preterm birth and stillbirth interventions will require addressing multiple barriers, and utilizing multiple delivery approaches and channels. Another important need is developing strategies to discontinue ineffective or harmful interventions. Preterm birth and stillbirth interventions must also be placed in the broader maternal, newborn and child health context to identify and prioritize those that will help improve several outcomes at the same time. The next article discusses advocacy challenges and opportunities.
doi:10.1186/1471-2393-10-S1-S4
PMCID: PMC2841777  PMID: 20233385
14.  Global report on preterm birth and stillbirth (6 of 7): ethical considerations 
BMC Pregnancy and Childbirth  2010;10(Suppl 1):S6.
Introduction
Despite the substantial global burden of preterm and stillbirth, little attention has been given to the ethical considerations related to research and interventions in the global context. Ethical dilemmas surrounding reproductive decisions and the care of preterm newborns impact the delivery of interventions, and are not well understood in low-resource settings. Issues such as how to address the moral and cultural attitudes surrounding stillbirths, have cross-cutting implications for global visibility of the disease burden. This analysis identifies ethical issues impacting definitions, discovery, development, and delivery of effective interventions to decrease the global burden of preterm birth and stillbirth.
Methods
This review is based on a comprehensive literature review; an ethical analysis of other articles within this global report; and discussions with GAPPS's Scientific Advisory Council, team of international investigators, and a community of international experts on maternal, newborn, and child health and bioethics from the 2009 International Conference on Prematurity and Stillbirth. The literature review includes articles in PubMed, Academic Search Complete (EBSCO), and Philosopher's Index with a range of 1995-2008.
Results
Advancements in discovery science relating to preterm birth and stillbirth require careful consideration in the design and use of repositories containing maternal specimens and data. Equally important is the need to improve clinical translation from basic science research to delivery of interventions, and to ensure global needs inform discovery science agenda-setting. Ethical issues in the development of interventions include a need to balance immediate versus long-term impacts—such as caring for preterm newborns rather than preventing preterm births. The delivery of interventions must address: women's health disparities as determinants of preterm birth and stillbirth; improving measurements of impact on equity in coverage; balancing maternal and newborn outcomes in choosing interventions; and understanding the personal and cross-cultural experiences of preterm birth and stillbirth among women, families and communities.
Conclusion
Efforts to improve visibility, funding, research and the successful delivery of interventions for preterm birth and stillbirth face a number of ethical concerns. Thoughtful input from those in health policy, bioethics and international research ethics helped shape an interdisciplinary global action agenda to prevent preterm birth and stillbirth.
doi:10.1186/1471-2393-10-S1-S6
PMCID: PMC2841776  PMID: 20233387
15.  Global report on preterm birth and stillbirth (7 of 7): mobilizing resources to accelerate innovative solutions (Global Action Agenda) 
BMC Pregnancy and Childbirth  2010;10(Suppl 1):S7.
Background
Preterm birth and stillbirth are complex local and global health problems requiring an interdisciplinary approach and an international commitment. Stakeholders developed recommendations for a Global Action Agenda (GAA) at the 2009 International Conference on Prematurity and Stillbirth. The primary goal of this GAA is to forge a collaborative effort toward achieving common goals to prevent preterm birth and stillbirth, and to improve related maternal, newborn, and child health outcomes.
Conference participants
GAPPS co-convened this four-day conference with the Bill & Melinda Gates Foundation, March of Dimes, PATH, Save the Children, UNICEF and the World Health Organization. Participants included about 200 leading international researchers, policymakers, health care practitioners and philanthropists. A near-final draft of this report was sent three weeks in advance to help co-chairs and participants prepare for workgroup discussions.
Global Action Agenda
Twelve thematic workgroups, composed of interdisciplinary experts, made recommendations on short-, intermediate-, and long-term milestones, and success metrics. Recommendations are based on the following themes: (1) advance discovery of the magnitude, causes and innovative solutions; (2) promote development and delivery of low-cost, proven interventions; (3) improve advocacy efforts to increase awareness that preterm birth and stillbirth are leading contributors to the global health burden; (4) increase resources for research and implementation; and (5) consider ethical and social justice implications throughout all efforts.
Summary
The conference provided an unprecedented opportunity for maternal, newborn and child health stakeholders to create a collaborative strategy for addressing preterm birth and stillbirth globally. Participants and others have already completed or launched work on key milestones identified in the GAA. Updates will be provided at www.gapps.org.
doi:10.1186/1471-2393-10-S1-S7
PMCID: PMC2841775  PMID: 20233388
16.  Global report on preterm birth and stillbirth (2 of 7): discovery science 
BMC Pregnancy and Childbirth  2010;10(Suppl 1):S2.
Background
Normal and abnormal processes of pregnancy and childbirth are poorly understood. This second article in a global report explains what is known about the etiologies of preterm births and stillbirths and identifies critical gaps in knowledge. Two important concepts emerge: the continuum of pregnancy, beginning at implantation and ending with uterine involution following birth; and the multifactorial etiologies of preterm birth and stillbirth. Improved tools and data will enable discovery scientists to identify causal pathways and cost-effective interventions.
Pregnancy and parturition continuum
The biological process of pregnancy and childbirth begins with implantation and, after birth, ends with the return of the uterus to its previous state. The majority of pregnancy is characterized by rapid uterine and fetal growth without contractions. Yet most research has addressed only uterine stimulation (labor) that accounts for <0.5% of pregnancy.
Etiologies
The etiologies of preterm birth and stillbirth differ by gestational age, genetics, and environmental factors. Approximately 30% of all preterm births are indicated for either maternal or fetal complications, such as maternal illness or fetal growth restriction. Commonly recognized pathways leading to preterm birth occur most often during the gestational ages indicated: (1) inflammation caused by infection (22-32 weeks); (2) decidual hemorrhage caused by uteroplacental thrombosis (early or late preterm birth); (3) stress (32-36 weeks); and (4) uterine overdistention, often caused by multiple fetuses (32-36 weeks). Other contributors include cervical insufficiency, smoking, and systemic infections. Many stillbirths have similar causes and mechanisms. About two-thirds of late fetal deaths occur during the antepartum period; the other third occur during childbirth. Intrapartum asphyxia is a leading cause of stillbirths in low- and middle-income countries.
Recommendations
Utilizing new systems biology tools, opportunities now exist for researchers to investigate various pathways important to normal and abnormal pregnancies. Improved access to quality data and biological specimens are critical to advancing discovery science. Phenotypes, standardized definitions, and uniform criteria for assessing preterm birth and stillbirth outcomes are other immediate research needs.
Conclusion
Preterm birth and stillbirth have multifactorial etiologies. More resources must be directed toward accelerating our understanding of these complex processes, and identifying upstream and cost-effective solutions that will improve these pregnancy outcomes.
doi:10.1186/1471-2393-10-S1-S2
PMCID: PMC2841774  PMID: 20233383
17.  Global report on preterm birth and stillbirth (5 of 7): advocacy barriers and opportunities 
BMC Pregnancy and Childbirth  2010;10(Suppl 1):S5.
Background
Efforts to achieve the Millennium Development Goals (MDGs) to improve maternal and child health can be accelerated by addressing preterm birth and stillbirth. However, most global health stakeholders are unaware of the inextricable connections of these adverse pregnancy outcomes to maternal, newborn and child health (MNCH). Improved visibility of preterm births and stillbirths will help fuel investments and strengthen commitments in the discovery, development and delivery of low-cost solutions globally. This article addresses potential barriers and opportunities to increasing global awareness and understanding.
Methods
Qualitative research was conducted to analyze current knowledge, attitudes and commitments toward preterm birth and stillbirth; identify advocacy challenges; and learn more about examples of programs that successfully advocate for research and appropriate interventions. Forty-one individuals from 14 countries on six continents were interviewed. They included maternal, newborn, and child health advocates and implementers, United Nations agency representatives, policymakers, researchers, and private and government donors.
Results
A common recognition of three advocacy challenges with regard to preterm birth and stillbirth emerged from these interviews: (1) lack of data about the magnitude and impact; (2) lack of awareness and understanding; and (3) lack of low-cost, effective and scalable interventions. Participants also identified advocacy opportunities. The first of these opportunities involves linking preterm birth and stillbirth to the MDGs, adding these outcomes to broader global health discussions and advocacy efforts, and presenting a united voice among advocates in the context of broader MNCH issues when addressing preterm birth and stillbirth. Another key opportunity is putting a human face to these tragedies—such as a parent who can speak to the personal impact on the family. Lastly, several interviewees suggested identifying and engaging champions to garner additional visibility and strengthen efforts. Ideal champions will work collaboratively with these and other maternal, newborn and child health issues. Conclusion: Advocacy efforts to add preterm births and stillbirths to broader MNCH goals, such as the MDGs, and to identify champions for these issues, will accelerate interdisciplinary efforts to reduce these adverse outcomes. The next article in this report presents an overview of related ethical considerations.
doi:10.1186/1471-2393-10-S1-S5
PMCID: PMC2841773  PMID: 20233386
18.  Global report on preterm birth and stillbirth (1 of 7): definitions, description of the burden and opportunities to improve data 
BMC Pregnancy and Childbirth  2010;10(Suppl 1):S1.
Introduction
This is the first of seven articles from a preterm birth and stillbirth report. Presented here is an overview of the burden, an assessment of the quality of current estimates, review of trends, and recommendations to improve data.
Preterm birth
Few countries have reliable national preterm birth prevalence data. Globally, an estimated 13 million babies are born before 37 completed weeks of gestation annually. Rates are generally highest in low- and middle-income countries, and increasing in some middle- and high-income countries, particularly the Americas. Preterm birth is the leading direct cause of neonatal death (27%); more than one million preterm newborns die annually. Preterm birth is also the dominant risk factor for neonatal mortality, particularly for deaths due to infections. Long-term impairment is an increasing issue.
Stillbirth
Stillbirths are currently not included in Millennium Development Goal tracking and remain invisible in global policies. For international comparisons, stillbirths include late fetal deaths weighing more than 1000g or occurring after 28 weeks gestation. Only about 2% of all stillbirths are counted through vital registration and global estimates are based on household surveys or modelling. Two global estimation exercises reached a similar estimate of around three million annually; 99% occur in low- and middle-income countries. One million stillbirths occur during birth. Global stillbirth cause-of-death estimates are impeded by multiple, complex classification systems.
Recommendations to improve data
(1) increase the capture and quality of pregnancy outcome data through household surveys, the main data source for countries with 75% of the global burden; (2) increase compliance with standard definitions of gestational age and stillbirth in routine data collection systems; (3) strengthen existing data collection mechanisms—especially vital registration and facility data—by instituting a standard death certificate for stillbirth and neonatal death linked to revised International Classification of Diseases coding; (4) validate a simple, standardized classification system for stillbirth cause-of-death; and (5) improve systems and tools to capture acute morbidity and long-term impairment outcomes following preterm birth.
Conclusion
Lack of adequate data hampers visibility, effective policies, and research. Immediate opportunities exist to improve data tracking and reduce the burden of preterm birth and stillbirth.
doi:10.1186/1471-2393-10-S1-S1
PMCID: PMC2841772  PMID: 20233382
19.  Global report on preterm birth and stillbirth (3 of 7): evidence for effectiveness of interventions 
BMC Pregnancy and Childbirth  2010;10(Suppl 1):S3.
Introduction
Interventions directed toward mothers before and during pregnancy and childbirth may help reduce preterm births and stillbirths. Survival of preterm newborns may also be improved with interventions given during these times or soon after birth. This comprehensive review assesses existing interventions for low- and middle-income countries (LMICs).
Methods
Approximately 2,000 intervention studies were systematically evaluated through December 31, 2008. They addressed preterm birth or low birth weight; stillbirth or perinatal mortality; and management of preterm newborns. Out of 82 identified interventions, 49 were relevant to LMICs and had reasonable amounts of evidence, and therefore selected for in-depth reviews. Each was classified and assessed by the quality of available evidence and its potential to treat or prevent preterm birth and stillbirth. Impacts on other maternal, fetal, newborn or child health outcomes were also considered. Assessments were based on an adaptation of the Grades of Recommendation Assessment, Development and Evaluation criteria.
Results
Most interventions require additional research to improve the quality of evidence. Others had little evidence of benefit and should be discontinued. The following are supported by moderate- to high-quality evidence and strongly recommended for LMICs:
• Two interventions prevent preterm births—smoking cessation and progesterone
• Eight interventions prevent stillbirths—balanced protein energy supplementation, screening and treatment of syphilis, intermittant presumptive treatment for malaria during pregnancy, insecticide-treated mosquito nets, birth preparedness, emergency obstetric care, cesarean section for breech presentation, and elective induction for post-term delivery
• Eleven interventions improve survival of preterm newborns—prophylactic steroids in preterm labor, antibiotics for PROM, vitamin K supplementation at delivery, case management of neonatal sepsis and pneumonia, delayed cord clamping, room air (vs. 100% oxygen) for resuscitation, hospital-based kangaroo mother care, early breastfeeding, thermal care, and surfactant therapy and application of continued distending pressure to the lungs for respiratory distress syndrome
Conclusion
The research paradigm for discovery science and intervention development must be balanced to address prevention as well as improve morbidity and mortality in all settings. This review also reveals significant gaps in current knowledge of interventions spanning the continuum of maternal and fetal outcomes, and the critical need to generate further high-quality evidence for promising interventions.
doi:10.1186/1471-2393-10-S1-S3
PMCID: PMC2841444  PMID: 20233384
20.  Staphylococcus aureus Elicits Marked Alterations in the Airway Proteome during Early Pneumonia▿ ‡  
Infection and Immunity  2008;76(12):5862-5872.
Pneumonia caused by Staphylococcus aureus is a growing concern in the health care community. We hypothesized that characterization of the early innate immune response to bacteria in the lungs would provide insight into the mechanisms used by the host to protect itself from infection. An adult mouse model of Staphylococcus aureus pneumonia was utilized to define the early events in the innate immune response and to assess the changes in the airway proteome during the first 6 h of pneumonia. S. aureus actively replicated in the lungs of mice inoculated intranasally under anesthesia to cause significant morbidity and mortality. By 6 h postinoculation, the release of proinflammatory cytokines caused effective recruitment of neutrophils to the airway. Neutrophil influx, loss of alveolar architecture, and consolidated pneumonia were observed histologically 6 h postinoculation. Bronchoalveolar lavage fluids from mice inoculated with phosphate-buffered saline (PBS) or S. aureus were depleted of overabundant proteins and subjected to strong cation exchange fractionation followed by liquid chromatography and tandem mass spectrometry to identify the proteins present in the airway. No significant changes in response to PBS inoculation or 30 min following S. aureus inoculation were observed. However, a dramatic increase in extracellular proteins was observed 6 h postinoculation with S. aureus, with the increase dominated by inflammatory and coagulation proteins. The data presented here provide a comprehensive evaluation of the rapid and vigorous innate immune response mounted in the host airway during the earliest stages of S. aureus pneumonia.
doi:10.1128/IAI.00865-08
PMCID: PMC2583584  PMID: 18852243
21.  Characterization of the Accessory Sec System of Staphylococcus aureus▿  
Journal of Bacteriology  2008;190(18):6188-6196.
The SraP adhesin of Staphylococcus aureus is a member of a highly conserved family of serine-rich surface glycoproteins of gram-positive bacteria. For streptococci, export of the SraP homologs requires a specialized transport pathway (the accessory Sec system). Compared to streptococci, however, SraP is predicted to differ in its signal peptide and glycosylation, which may affect its dependence on a specialized system for transport. In addition, two genes (asp4 and asp5) essential for export in Streptococcus gordonii are missing in S. aureus. Thus, the selectivity of the accessory Sec system in S. aureus may also differ compared to streptococci. To address these issues, the five genes encoding the putative accessory Sec system (secY2, secA2, and asp1-3) were disrupted individually in S. aureus ISP479C, and the resultant mutants were examined for SraP export. Disruption of secA2 resulted in the near complete loss of SraP surface expression. Similar results were seen with disruption of secY2 and asp1, asp2, or asp3. To assess whether the accessory Sec system transported other substrates, we compared secreted proteomes of ISP479C and a secA2 isogenic mutant, by two-dimensional fluorescence difference gel electrophoresis. Although two consistent differences in proteome content were noted between the strains, neither protein appeared to be a likely substrate for accessory Sec export. Thus, the accessory Sec system of S. aureus is required for the export of SraP, and it appears to be dedicated to the transport of this substrate exclusively.
doi:10.1128/JB.00300-08
PMCID: PMC2546797  PMID: 18621893
22.  Regulation of cytotoxin expression by converging eukaryotic-type and two-component signalling mechanisms in Streptococcus agalactiae 
Molecular microbiology  2006;62(4):941-957.
Summary
Signal transducing mechanisms are essential for regulation of gene expression in both prokaryotic and eukaryotic organisms. Regulation of gene expression in eukaryotes is accomplished by serine/threonine and tyrosine kinases and cognate phosphatases. In contrast, gene expression in prokaryotes is controlled by two-component systems that comprise a sensor histidine kinase and a cognate DNA binding response regulator. Pathogenic bacteria utilize two-component systems to regulate expression of their virulence factors and for adaptive responses to the external environment. We have previously shown that the human pathogen Streptococcus agalactiae (Group B Streptococci, GBS) encodes a single eukaryotic-type serine/threonine kinase Stk1, which is important for virulence of the organism. In this study, we aimed to understand how Stk1 contributes to virulence of GBS. Our results indicate that Stk1 expression is important for resistance of GBS to human blood, neutrophils and oxidative stress. Consistent with these observations, Stk1 positively regulates transcription of a cytotoxin, β-haemolysin/cytolysin (β-H/C) that is critical for survival of GBS in the bloodstream and for resistance to oxidative stress. Interestingly, positive regulation of β-H/C by Stk1 requires the two-component regulator CovR. Further, we show that Stk1 can negatively regulate transcription of CAMP factor in a CovR-dependent manner. As Stk1 phosphorylates CovR in vitro, these data suggest that serine/threonine phosphorylation impacts CovR-mediated regulation of GBS gene expression. In summary, our studies provide novel information that a eukaryotic-type serine/threonine kinase regulates two-component-mediated expression of GBS cytotoxins.
doi:10.1111/j.1365-2958.2006.05431.x
PMCID: PMC2593684  PMID: 17005013
23.  Crystallization and preliminary crystallographic analysis of two Streptococcus agalactiae proteins: the family II inorganic pyrophosphatase and the serine/threonine phosphatase 
Two S. agalactiae proteins, the inorganic pyrophosphatase and the serine/threonine phosphatase, were crystallized and diffraction data were collected and processed from these crystals. The data from the two protein crystals extended to 2.80 and 2.65 Å, respectively.
Streptococcus agalactiae, which infects human neonates and causes sepsis and meningitis, has recently been shown to possess a eukaryotic-like serine/threonine protein phosphorylation signalling cascade. Through their target proteins, the S. agalactiae Ser/Thr kinase and Ser/Thr phosphatase together control the growth as well as the morphology and virulence of this organism. One of the targets is the S. agalactiae family II inorganic pyrophosphatase. The inorganic pyrophosphatase and the serine/threonine phosphatase have therefore been purified and crystallized and diffraction data have been collected from their crystals. The data were processed using XDS. The inorganic pyrosphosphatase crystals diffracted to 2.80 Å and the Ser/Thr phosphatase crystals to 2.65 Å. Initial structure-solution experiments indicate that structure solution will be successful in both cases. Solving the structure of the proteins involved in this cascade is the first step towards understanding this phenomenon in atomic detail.
doi:10.1107/S174430910602954X
PMCID: PMC2242878  PMID: 16946472
Streptococcus agalactiae; inorganic pyrophosphatase; serine/threonine phosphatase
24.  Host Airway Proteins Interact with Staphylococcus aureus during Early Pneumonia▿ †  
Infection and Immunity  2008;76(3):888-898.
Staphylococcus aureus is a major cause of hospital-acquired pneumonia and is emerging as an important etiological agent of community-acquired pneumonia. Little is known about the specific host-pathogen interactions that occur when S. aureus first enters the airway. A shotgun proteomics approach was utilized to identify the airway proteins associated with S. aureus during the first 6 h of infection. Host proteins eluted from bacteria recovered from the airways of mice 30 min or 6 h following intranasal inoculation under anesthesia were subjected to liquid chromatography and tandem mass spectrometry. A total of 513 host proteins were associated with S. aureus 30 min and/or 6 h postinoculation. A majority of the identified proteins were host cytosolic proteins, suggesting that S. aureus was rapidly internalized by phagocytes in the airway and that significant host cell lysis occurred during early infection. In addition, extracellular matrix and secreted proteins, including fibronectin, antimicrobial peptides, and complement components, were associated with S. aureus at both time points. The interaction of 12 host proteins shown to bind to S. aureus in vitro was demonstrated in vivo for the first time. The association of hemoglobin, which is thought to be the primary staphylococcal iron source during infection, with S. aureus in the airway was validated by immunoblotting. Thus, we used our recently developed S. aureus pneumonia model and shotgun proteomics to validate previous in vitro findings and to identify nearly 500 other proteins that interact with S. aureus in vivo. The data presented here provide novel insights into the host-pathogen interactions that occur when S. aureus enters the airway.
doi:10.1128/IAI.01301-07
PMCID: PMC2258841  PMID: 18195024
25.  Structure of the Streptococcus agalactiae family II inorganic pyrophosphatase at 2.80 Å resolution 
Streptococcus agalactiae, a prokaryote that causes infections in neonates and immunocompromised adults, has a serine/threonine protein kinase (STK) signalling cascade. The structure of one of the targets, a family II inorganic pyrophosphatase, has been solved by molecular replacement and refined at 2.80 Å resolution to an R factor of 19.2% (Rfree = 26.7%). The two monomers in the asymmetric unit are related by a noncrystallographic twofold axis, but the biological dimer is formed by a crystallographic twofold. Each monomer contains the pyrophosphate analogue imidodiphosphate (PNP) and three metal ions per active site: two Mn2+ ions in sites M1 and M2 and an Mg2+ ion in site M3. The enzyme is in the closed conformation. Like other family II enzymes, the structure consists of two domains (residues 1–191 and 198–311), with the active site located between them. The conformation of Lys298 in the active site is different from those observed previously and it coordinates to the conserved DHH motif in a unique way. The structure suggests that Ser150, Ser194, Ser195 and Ser296 are the most likely targets for the Ser/Thr kinase and phosphatase because they are surface-accessible and either in the active site or in the hinge region between the two domains.
doi:10.1107/S0907444907019695
PMCID: PMC2365889  PMID: 17505113

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