Regulatory Requirements for Staphylococcus aureus Nitric Oxide Resistance

ABSTRACT The ability of Staphylococcus aureus to resist host innate immunity augments the severity and pervasiveness of its pathogenesis. Nitric oxide (NO˙) is an innate immune radical that is critical for the efficient clearance of a wide range of microbial pathogens. Exposure of microbes to NO˙ typically results in growth inhibition and induction of stress regulons. S. aureus , however, induces a metabolic state in response to NO˙ that allows for continued replication and precludes stress regulon induction. The regulatory factors mediating this distinctive response remain largely undefined. Here, we employ a targeted transposon screen and transcriptomics to identify and characterize five regulons essential for NO˙ resistance in S. aureus : three virulence regulons not formerly associated with NO˙ resistance, SarA, CodY, and Rot, as well as two regulons with established roles, Fur and SrrAB. We provide new insights into the contributions of Fur and SrrAB during NO˙ stress and show that the S. aureus Δ sarA mutant, the most sensitive of the newly identified mutants, exhibits metabolic dysfunction and widespread transcriptional dysregulation following NO˙ exposure. Altogether, our results broadly characterize the regulatory requirements for NO˙ resistance in S. aureus and suggest an intriguing overlap between the regulation of NO˙ resistance and virulence in this well-adapted human pathogen. IMPORTANCE The prolific human pathogen Staphylococcus aureus is uniquely capable of resisting the antimicrobial radical nitric oxide (NO˙), a crucial component of the innate immune response. However, a complete understanding of how S. aureus regulates an effective response to NO˙ is lacking. Here, we implicate three central virulence regulators, SarA, CodY, and Rot, as major players in the S. aureus NO˙ response. Additionally, we elaborate on the contribution of two regulators, SrrAB and Fur, already known to play a crucial role in S. aureus NO˙ resistance. Our study sheds light on a unique facet of S. aureus pathogenicity and demonstrates that the transcriptional response of S. aureus to NO˙ is highly pleiotropic and intrinsically tied to metabolism and virulence regulation

Biosensor Integration Development ExMC/Canadian Space Agency Collaboration

In support of the NASA Human Research Program Exploration Medical Capability (ExMC) Element, NASA Ames Research Center (ARC) established a collaborative effort with the Canadian Space Agency (CSA). The collaboration focuses on leveraging CSA capability in the areas of biosensors and decision support that will augment future development of such components for Exploration Missions. The CSA advancement of biosensors enables NASA to focus on the integration and data management associated with these types of components through the system currently under development by the Medical Data Architecture (MDA) project. This approach has enabled the establishment of a successful collaborative working relationship between ExMC and CSA.Applying lessons learned from the fiscal year 2016 (FY16) Human Exploration Research Analog (HERA) campaign, CSA and NASA ARC developed a solution to provide real-time feedback to researchers who monitor the collection of vital signs data from a wearable Astroskin garment. The advances in the interfaces included the development of an iPad application (by CSA) to wirelessly forward the vital signs data to the MDA system, which collected the vital signs data through a receiver developed by NASA ARC. The development of these interfaces aims to provide communications between the Astroskin and the MDA system such that data may be seamlessly collected, stored and retrieved by the MDA. The first steps towards this goal were demonstrated in FY16. In FY17, ExMC will complete the first in a series of test beds that establishes a system to automate collection and management of vital sign data from the Astroskin, and other sources of data, to provide information for a crewmember to make medical decisions. In addition, the MDA Test Bed 1 will enable CSA to evaluate and optimize biosensor advancement and facilitate decision support algorithm development

Epigenetic Tailoring for the Production of Anti-Infective Cytosporones from the Marine Fungus Leucostoma persoonii

Recent genomic studies have demonstrated that fungi can possess gene clusters encoding for the production of previously unobserved secondary metabolites. Activation of these attenuated or silenced genes to obtain either improved titers of known compounds or new ones altogether has been a subject of considerable interest. In our efforts to discover new chemotypes that are effective against infectious diseases, including malaria and methicillin-resistant Staphylococcus aureus (MRSA), we have isolated a strain of marine fungus, Leucostoma persoonii, that produces bioactive cytosporones. Epigenetic modifiers employed to activate secondary metabolite genes resulted in enhanced production of known cytosporones B (1, 360%), C (2, 580%) and E (3, 890%), as well as the production of the previously undescribed cytosporone R (4). Cytosporone E was the most bioactive, displaying an IC90 of 13 µM toward Plasmodium falciparum, with A549 cytotoxicity IC90 of 437 µM, representing a 90% inhibition therapeutic index (TI90 = IC90 A459/IC90 P. falciparum) of 33. In addition, cytosporone E was active against MRSA with a minimal inhibitory concentration (MIC) of 72 µM and inhibition of MRSA biofilm at roughly half that value (minimum biofilm eradication counts, MBEC90, was found to be 39 µM)

Identification and Characterization of σS, a Novel Component of the Staphylococcus aureus Stress and Virulence Responses

S. aureus is a highly successful pathogen that is speculated to be the most common cause of human disease. The progression of disease in S. aureus is subject to multi-factorial regulation, in response to the environments encountered during growth. This adaptive nature is thought to be central to pathogenesis, and is the result of multiple regulatory mechanisms employed in gene regulation. In this work we describe the existence of a novel S. aureus regulator, an as yet uncharacterized ECF-sigma factor (σS), that appears to be an important component of the stress and pathogenic responses of this organism. Using biochemical approaches we have shown that σS is able to associates with core-RNAP, and initiate transcription from its own coding region. Using a mutant strain we determined that σS is important for S. aureus survival during starvation, extended exposure to elevated growth temperatures, and Triton X-100 induced lysis. Coculture studies reveal that a σS mutant is significantly outcompeted by its parental strain, which is only exacerbated during prolonged growth (7 days), or in the presence of stressor compounds. Interestingly, transcriptional analysis determined that under standard conditions, S. aureus SH1000 does not initiate expression of sigS. Assays performed hourly for 72h revealed expression in typically background ranges. Analysis of a potential anti-sigma factor, encoded downstream of sigS, revealed it to have no obvious role in the upregulation of sigS expression. Using a murine model of septic arthritis, sigS-mutant infected animals lost significantly less weight, developed septic arthritis at significantly lower levels, and had increased survival rates. Studies of mounted immune responses reveal that sigS-mutant infected animals had significantly lower levels of IL-6, indicating only a weak immunological response. Finally, strains of S. aureus lacking sigS were far less able to undergo systemic dissemination, as determined by bacterial loads in the kidneys of infected animals. These results establish that σS is an important component in S. aureus fitness, and in its adaptation to stress. Additionally it appears to have a significant role in its pathogenic nature, and likely represents a key component in the S. aureus regulatory network

The novel two-component system AmsSR governs alternative metabolic pathway usage in Acinetobacter baumannii

In this study, we identify a novel two-component system in Acinetobacter baumannii (herein named AmsSR for regulator of alternative metabolic systems) only present in select gammaproteobacterial and betaproteobacterial species. Bioinformatic analysis revealed that the histidine kinase, AmsS, contains 14 predicted N-terminal transmembrane domains and harbors a hybrid histidine kinase arrangement in its C-terminus. Transcriptional analysis revealed the proton ionophore CCCP selectively induces P amsSR expression. Disruption of amsSR resulted in decreased intracellular pH and increased depolarization of cytoplasmic membranes. Transcriptome profiling revealed a major reordering of metabolic circuits upon amsR disruption, with energy generation pathways typically used by bacteria growing in limited oxygen being favored. Interestingly, we observed enhanced growth rates for mutant strains in the presence of glucose, which led to overproduction of pyruvate. To mitigate the toxic effects of carbon overflow, we noted acetate overproduction in amsSR-null strains, resulting from a hyperactive Pta-AckA pathway. Additionally, due to altered expression of key metabolic genes, amsSR mutants favor an incomplete TCA cycle, relying heavily on an overactive glyoxylate shunt. This metabolic reordering overproduces NADH, which is not oxidized by the ETC; components of which were significantly downregulated upon amsSR disruption. As a result, the mutants almost exclusively rely on substrate phosphorylation for ATP production, and consequently display reduced oxygen consumption in the presence of glucose. Collectively, our data suggests that disruption of amsSR affects the function of the aerobic respiratory chain, impacting the energy status of the cell, which in turn upregulates alternative metabolic and energy generation pathways

Persistence of Supplemented Bifidobacterium longum subsp. infantis EVC001 in Breastfed Infants.

Attempts to alter intestinal dysbiosis via administration of probiotics have consistently shown that colonization with the administered microbes is transient. This study sought to determine whether provision of an initial course of Bifidobacterium longum subsp. infantis (B. infantis) would lead to persistent colonization of the probiotic organism in breastfed infants. Mothers intending to breastfeed were recruited and provided with lactation support. One group of mothers fed B. infantis EVC001 to their infants from day 7 to day 28 of life (n = 34), and the second group did not administer any probiotic (n = 32). Fecal samples were collected during the first 60 postnatal days in both groups. Fecal samples were assessed by 16S rRNA gene sequencing, quantitative PCR, mass spectrometry, and endotoxin measurement. B. infantis-fed infants had significantly higher populations of fecal Bifidobacteriaceae, in particular B. infantis, while EVC001 was fed, and this difference persisted more than 30 days after EVC001 supplementation ceased. Fecal milk oligosaccharides were significantly lower in B. infantis EVC001-fed infants, demonstrating higher consumption of human milk oligosaccharides by B. infantis EVC001. Concentrations of acetate and lactate were significantly higher and fecal pH was significantly lower in infants fed EVC001, demonstrating alterations in intestinal fermentation. Infants colonized by Bifidobacteriaceae at high levels had 4-fold-lower fecal endotoxin levels, consistent with observed lower levels of Gram-negative Proteobacteria and Bacteroidetes. IMPORTANCE The gut microbiome in early life plays an important role for long-term health and is shaped in large part by diet. Probiotics may contribute to improvements in health, but they have not been shown to alter the community composition of the gut microbiome. Here, we found that breastfed infants could be stably colonized at high levels by provision of B. infantis EVC001, with significant changes to the overall microbiome composition persisting more than a month later, whether the infants were born vaginally or by caesarean section. This observation is consistent with previous studies demonstrating the capacity of this subspecies to utilize human milk glycans as a nutrient and underscores the importance of pairing a probiotic organism with a specific substrate. Colonization by B. infantis EVC001 resulted in significant changes to fecal microbiome composition and was associated with improvements in fecal biochemistry. The combination of human milk and an infant-associated Bifidobacterium sp. shows, for the first time, that durable changes to the human gut microbiome are possible and are associated with improved gut function

Contribution of the nos-pdt Operon to Virulence Phenotypes in Methicillin-Sensitive Staphylococcus aureus

Nitric oxide (NO) is emerging as an important regulator of bacterial stress resistance, biofilm development, and virulence. One potential source of endogenous NO production in the pathogen Staphylococcus aureus is its NO-synthase (saNOS) enzyme, encoded by the nos gene. Although a role for saNOS in oxidative stress resistance, antibiotic resistance, and virulence has been recently-described, insights into the regulation of nos expression and saNOS enzyme activity remain elusive. To this end, transcriptional analysis of the nos gene in S. aureus strain UAMS-1 was performed, which revealed that nos expression increases during low-oxygen growth and is growth-phase dependent. Furthermore, nos is co-transcribed with a downstream gene, designated pdt, which encodes a prephenate dehydratase (PDT) enzyme involved in phenylalanine biosynthesis. Deletion of pdt significantly impaired the ability of UAMS-1 to grow in chemically-defined media lacking phenylalanine, confirming the function of this enzyme. Bioinformatics analysis revealed that the operon organization of nos-pdt appears to be unique to the staphylococci. As described for other S. aureus nos mutants, inactivation of nos in UAMS-1 conferred sensitivity to oxidative stress, while deletion of pdt did not affect this phenotype. The nos mutant also displayed reduced virulence in a murine sepsis infection model, and increased carotenoid pigmentation when cultured on agar plates, both previously-undescribed nos mutant phenotypes. Utilizing the fluorescent stain 4-Amino-5-Methylamino-2',7'-Difluorofluorescein (DAF-FM) diacetate, decreased levels of intracellular NO/reactive nitrogen species (RNS) were detected in the nos mutant on agar plates. These results reinforce the important role of saNOS in S. aureus physiology and virulence, and have identified an in vitro growth condition under which saNOS activity appears to be upregulated. However, the significance of the operon organization of nos-pdt and potential relationship between these two enzymes remains to be elucidated

Medical Data Architecture Capabilities and Design

Mission constraints will challenge the delivery of medical care on a long-term, deep space explorationmission. This type of mission will be restricted in the availability of medical knowledge, skills, procedures and resourcesto prevent, diagnose, and treat in-flight medical events. Challenges to providing medical care are anticipated, includingresource and resupply constraints, delayed communications and no ability for medical evacuation. The Medical DataArchitecture (MDA) project will enable medical care capability in this constrained environment.The first version of thesystem, called Test Bed 1, includes capabilities for automated data collection, data storage and data retrieval to provideinformation to the Crew Medical Officer (CMO). Test Bed 1 seeks to establish a data architecture foundation and developa scalable data management system through modular design and standardized interfaces. In addition, it will demonstrateto stakeholders the potential for an improved, automated, flow of data to and from the medical system over the currentmethods employed on the International Space Station (ISS). It integrates a set of external devices, software andprocesses, and a Subjective, Objective, Assessment, and Plan (SOAP) note commonly used by clinicians. Medical datalike electrocardiogram plots, heart rate, skin temperature, respiration rate, medications taken, and more are collectedfrom devices and stored in the Electronic Medical Records (EMR) system, and reported to crew and clinician. Devicesintegrated include the Astroskin biosensor vest and IMED CARDIAX electrocardiogram (ECG) device with INEED MDECG Glove, and the NASA-developed Medical Dose Tracker application.The system is designed to be operated as astandalone system, and can be deployed in a variety of environments, from a laptop to a data center. The system isprimarily composed of open-source software tools, and is designed to be modular, so new capabilities can be added. Thesoftware components and integration methods will be discussed

Medical Data Architecture Project Status

The Medical Data Architecture (MDA) project supports the Exploration Medical Capability (ExMC) risk to minimize or reduce the risk of adverse health outcomes and decrements in performance due to in-flight medical capabilities on human exploration missions. To mitigate this risk, the ExMC MDA project addresses the technical limitations identified in ExMC Gap Med 07: We do not have the capability to comprehensively process medically-relevant information to support medical operations during exploration missions. This gap identifies that the current International Space Station (ISS) medical data management includes a combination of data collection and distribution methods that are minimally integrated with on-board medical devices and systems. Furthermore, there are variety of data sources and methods of data collection. For an exploration mission, the seamless management of such data will enable an increasingly autonomous crew than the current ISS paradigm. The MDA will develop capabilities that support automated data collection, and the necessary functionality and challenges in executing a self-contained medical system that approaches crew health care delivery without assistance from ground support.To attain this goal, the first year of the MDA project focused on reducing technical risk, developing documentation and instituting iterative development processes that established the basis for the first version of MDA software (or Test Bed 1). Test Bed 1 is based on a nominal operations scenario authored by the ExMC Element Scientist. This narrative was decomposed into a Concept of Operations that formed the basis for Test Bed 1 requirements. These requirements were successfully vetted through the MDA Test Bed 1 System Requirements Review, which permitted the MDA project to begin software code development and component integration. This paper highlights the MDA objectives, development processes, and accomplishments, and identifies the fiscal year 2017 milestones and deliverables in the upcoming year

Multigenomic Delineation of Plasmodium Species of the Laverania Subgenus Infecting Wild-living Chimpanzees and Gorillas

Plasmodium falciparum, the major cause of malaria morbidity and mortality worldwide, is only distantly related to other human malaria parasites and has thus been placed in a separate subgenus, termed Laverania. Parasites morphologically similar to P. falciparum have been identified in African apes, but only one other Laverania species, Plasmodium reichenowi from chimpanzees, has been formally described. Although recent studies have pointed to the existence of additional Laverania species, their precise number and host associations remain uncertain, primarily because of limited sampling and a paucity of parasite sequences other than from mitochondrial DNA. To address this, we used limiting dilution polymerase chain reaction to amplify additional parasite sequences from a large number of chimpanzee and gorilla blood and fecal samples collected at two sanctuaries and 30 field sites across equatorial Africa. Phylogenetic analyses of more than 2,000 new sequences derived from the mitochondrial, nuclear, and apicoplast genomes revealed six divergent and well-supported clades within the Laverania parasite group. Although two of these clades exhibited deep subdivisions in phylogenies estimated from organelle gene sequences, these sublineages were geographically defined and not present in trees from four unlinked nuclear loci. This greatly expanded sequence data set thus confirms six, and not seven or more, ape Laverania species, of which P. reichenowi, Plasmodium gaboni, and Plasmodium billcollinsi only infect chimpanzees, whereas Plasmodium praefalciparum, Plasmodium adleri, and Pladmodium blacklocki only infect gorillas. The new sequence data also confirm the P. praefalciparum origin of human P. falciparum