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Focus Equally on the Pathogens .....
||.... and on the Host Response to Infection|
Group A Streptococcus (GAS) is an important human pathogen causing diseases ranging from simple pharyngitis ("strep throat") to invasive necrotizing fasciitis ("flesh-eating disease") to the immune-mediated syndrome of rheumatic fever. Our lab aims to discover and characterize GAS virulence factors by coupling genetic approaches (e.g. targeted mutagenesis) with tissue culture and murine infection models. Of particular interest are genetic mechanisms of GAS innate immune resistance and the shift from mucosal colonization to systemic disease. Among the GAS virulence factors we study are the pore-forming toxins streptolysins S and O (SLS and SLO), the antiphagocytic and proinflammatory surface M protein, DNAse Sda1, cysteine protease SpeB, hyaluronic acid capsule, serum opacity factor, IL-8 peptidase, and the cell wall group A carbohydrate. Together, these studies aim to provide new targets for drug therapy and vaccine prophylaxis. Our collaborators include M. Walker (U. Queensland), P. Ghosh (UCSD), J. Dixon (UCSD), M. Kotb (Cincinnati) and E. Hanski (Jerusalem).
Streptococcus (GBS) is the leading cause of invasive
bacterial infections in human newborn infants, including
sepsis and meningitis. GBS is also increasingly associated
infections in nonpregnant adults, especially those with
diseases that weaken immunity. Our laboratory has studied
aspects of GBS pathogenesis through random transposon and
exchange mutagenesis paired with in
vitro cell culture models and
in vivo small animal challenges. Major areas of
have include the molecular genetics and virulence
properties of the GBS
pore-forming hemolysin/cytolysin toxin, functions of the
acid-expressing GBS polysaccharide capsule in molecular
evasion of innate immune clearance, mechanisms of lung
inflammatory responses in GBS pneumonia in the premature
role of GBS surface proteins and cell wall components in
adherence and invasion, and the molecular basis of GBS
and injury to the blood-brain barrier endothelium in the
of newborn meningitis. Our collaborators include
(SDSU) and Ajit Varki (UCSD).
| Staphylococcus aureus
Staphylococcus aureus causes nosocomial and community-acquired diseases including skin and soft tissue infections, osteomyelitis, bacteremia, abscesses, endocarditis and septicemia. Antibiotic resistance has reached epidemic proportions in many regions, with methicillin-resistant S. aureus (MRSA) now exceeding HIV/AIDS as a cause of death in the U.S. Our S. aureus research includes study of how the pathogen resists killing by human phagocytes, including the antioxidant properties of its golden carotenoid pigment, staphyloxanthin. Pigment inhibition, achieved through repurposing of human cholesterol-lowering agents, may hold promise as an adjunct to antibiotic therapy of MRSA. Other immune evasion factors under investigation include S. aureus nitric oxide synthase, phenol soluble modulins, Ig-binding protein A and the pore-forming α-hemolysin. We are also investigating S. aureus colonization and skin infection, to better understand how the pathogen both activates and resists cutaneous innate defenses. Our collaborators include George Liu (Cedars-Sinai), Eric Oldfield (U. Illinois), Pieter Dorrestein (UCSD), Suzan Roiijakkers (Utrecht) and AuricX Pharmaceuticals (Houston).
|Streptococcus pneumoniae (SPN) is perhaps the leading cause of clinically significant bacterial infections worldwide, with a disease spectrum ranging from simple otitis media and sinusitis to invasive conditions including pneumonia, sepsis and meningitis. We have been interested in multiple roles of the surface-anchored pneumococcal neuraminidase (sialidase), NanA, in disease pathogenesis. In addition to its ability to cleave terminal sialic acid motifs on host cell targets, we have shown that an additional domain of the protein promotes invasion of brain microvascular endothelial cells and the development of pneumococal meningitis. Also, by cleaving sialic acid from host cell surfaces, the tonic engagement in cis of inhibitory Siglec receptors is released, leading to exaggerated leukocyte inflammatory responses. Finally, we have studied how NanactivitiesA sialidase modifies platelets and clotting factors to promote their clearance by the hepatic Ashwell receptor, influencing the development of disseminated intravascular coagulation during pneumococcal sepsis. Our collaborators include Ajit Varki (UCSD), Jamey Marth (SBMRI), and Kelly Doran (SDSU).||Pneumococcal
| Bacillus anthracis
Bacillus anthracis is a Gram-positive spore-forming bacterium and the causative agent of anthrax. Primarily a disease of livestock, anthrax can infect humans through cutaneous, respiratory or gastrointestinal routes of infection. Inhalational anthrax occurs when endospores are introduced to the lung and taken up by resident phagocytes -- the high lethality of this disease makes anthrax a foremost biodefense concern, as evidenced by the 2001 postal attacks. We are studying novel functions of the anthrax toxins edema factor (EF) and lethal factor (LF) to inhibit endocytic recycling by the Rab11/Sec15 exocyst, leading to disruption of tight junctions and cell barriers, a finding we have generalized to other cAMP-inducing toxins. We are also studying the survival response of macrophages to LF inhibition of p38 MAPK, a pathway that involves activation of NOD2-dependent inflammasomes via ATP release and adenosine receptor signaling. Finally, we are looking at novel antimicrobial peptide resistance factors such as the ClpX protease which promote anthrax innate immune resistance and virulence. Collaborators include Ethan Bier (UCSD), Michael Karin (UCSD) and Shauna McGillivray (TCU).
|Streptococcus iniae infections in aquaculture The controlled aquaculture of fish is an important and cost-effective industry for increasing the world's food supply. However, intensive aquaculture of several fish species has been threatened by infectious diseases, in particular a fatal meningoencephalitis produced by the ▀-hemolytic Streptococcus iniae. In recent years, we have conducted collaborative research project seeks to elucidate the virulence mechanisms of S. iniae using molecular techniques of random and targeted mutagenesis together with in vivo screening assays for virulence potential in hybrid-striped bass, tilapia and other species. Our goal is the rational development of effect vaccines and novel therapeutic strategies to protect aquacultured fish against this often devastating pathogen. A byproduct of this research has been participation in the discovery and characterization of novel fish antimicrobial peptides (e.g. moronecidin, bass hepcidin) and elucidation of their role in fish innate immune defense.||Streptococcus iniae
| Cathelicidin AMPs in Skin
Cathelicidin antimicrobial peptides (AMPs) In a complex environment, higher organisms face the constant threat of microbial infection. Effective first lines of defense against infectious pathogens comprise the innate immune system. A key component of innate immunity is the production of small, cationic AMPs, a protection strategy conserved from insects through man. In a longstanding collaboration with the laboratory of Richard Gallo (UCSD), we have adopted a combined mammalian and bacterial genetic approach to decipher the contributions of the cathelicidin family of AMPs to host immunity. Our studies with cathelicidin KO mice and GAS infection provided the first in vivo demonstration that endogenous expression of a mammalian antimicrobial peptide protects against invasive bacterial infection. Ongoing collaborative projects now explore additional immunostimulatory functions of the cathelicidin molecule, its transcriptional regulation by HIF and VitD in response to infectious challenge, the molecular and phenotypic basis of bacterial sensitivity or resistance to AMP action, and the impact of bacterial AMP resistance on virulence and infectious disease epidemiology.
|Hypoxia-inducible factor (HIF-1) Through a longstanding collaboration with the group of Randall Johnson (Cambridge and UCSD), we are examining the role of transcription factor HIF-1α as a key regulator of the bactericidal and inflammatory capacity of macrophages and neutrophils. Hypoxia is a characteristic feature of the tissue microenvironment during bacterial infection. HIF-1α is induced by infection, even under normoxia, and regulates the production of key immune effector molecules including granule proteases, antimicrobial peptides, nitric oxide and TNFα. Mice lacking HIF-1α in their myeloid cell lineage show decreased bactericidal activity and failed to restrict systemic spread of infection from an initial tissue focus. Conversely, activation of the HIF-1α pathway through vHL deletion or pharmacologic inducers supports myeloid cell production of defense factors and improved bactericidal capacity. Drug development for treatment of difficult infectious diseases is pursued in collaboration with Aerpio Therapeutics (Cincinnati). We are also examining the role of HIF-1α in modulating airway inflammation in asthma with L. Crotty Alexander (UCSD).||HIF-1 and
Glycobiology of host-pathogen interactions. The surface of all bacterial and human cells are covered with glycan molecules that play a primary role in arbitrating the outcome of the host-pathogen encounter. We are members of the UCSD Program in Excellence in Glycosciences (PEG) and Glycobiology Research and Training Center (GRTC) spearheading collaborative projects that examine the role of bacterial glycans and glycosidases in modulating myeloid cell innate immune and inflammatory responses. One major focus is the role of sialic acid binding lectins known as Siglecs in regulation of leukocyte function, and their subversion through molecular mimicry by GBS expressing its sialylated polysaccharide capsule. We are also studying the role of GAS hyaluronic acid in interactions with CD44, proinflammatory effects of bacterial sialidases and hyaluronidases, the role of host glycosaminoglycans in neutrophil and endothelial barrier function during infection, and the potential of reprogramming natural antibodies against the nonhuman αGal epitope to clear drug-resistant pathogens via engineered RNA aptamers. Collaborators include A. Varki (UCSD), J. Esko (UCSD), R. Gallo (UCSD) and Altermune Technologies (London).
|Immune signaling in macrophages. In addition to our work with HIF-1 and Siglecs described above, we are probing a variety of signal transduction mechanisms and cellular pathways by which macrophages are rapidly activated in response to bacterial infection, but then able to resolve inflammation to limit collateral damage to host tissues. Areas of investigation include the roles of IKK/NFkB and MAP kinase pathways in regulating macrophage bactericidal and cytokine responses to infection, the function of NOD-like intracellular pattern receptors in response to bacterial toxins, mechanisms of inflammasome activation and IL-1▀ signaling, bacterial modulation of host cell apoptotic pathways, ATP/adenosine receptor signaling in neutrophil chemotaxis and bacterial killing, and the role of autophagy in host defense against intracellular pathogens. As a new participant of the NIH/NIAID Great Lakes Regional Center for Excellence in Biodefense and Emerging Infectious Disease Research, we are utilizing the information gained from these studies to design strategies for pharmacological enhancement of phagocytic cell function vs. antibiotic-resistant pathogens. Collaborators include Michael Karin (UCSD), Christopher Glass (UCSD) and Zev Ronai (SBMRI).||Immune Signaling
| Neutrophil Extracellular
Neutrophil extracellular traps (ETs) consist of nuclear (or mitochondrial) DNA as a backbone with embedded antimicrobial peptides, histones, and cell-specific proteases providing a matrix to entrap and kill microbes . NETs are formed after stimulation with mitogens, cytokines, or pathogens themselves, in a specialized cell death process involving a ROS-mediated signaling cascade and particular chromatin modifications. We are exploring roles of HIF-1α and cathelicidins in the generation of NETs at peripheral foci of infection, and have uncovered a novel contribution of the cholesterol biosynthetic pathway in the regulation of NET formation. We are also investigating other immune cells including mast cells and macrophages can themselves produce extracellular traps to control pathogens. Companion projects examine how specific bacterial factors (e.g. GAS M protein) stimulate NET production, whereas other virulence factors promote bacterial resistance to NET killing, e.g. by degradation of the NET architecture (DNAses of GAS and S. aureus) or resistance to the embedded cathelicidins. Collaborators include Maren von K÷ckritz-Blickwede (Hannover).
|Novel antibiotic discovery. The continual emergence of antibiotic resistance among medically-important bacterial pathogens poses a great challenge to the public health. Sadly, the pipeline of new antibiotics in pharmaceutical development has yet to match this threat, with few novel antibiotic scaffolds developed in the last few decades. Our group is pursuing multiple parallel approaches for novel antibiotic discovery. These include evaluation of new chemical entities generated from marine actinomycete-derived natural product libraries, chemical genomic platforms, virtual screens, and medical chemistry modification of lead compounds. We also believe that outside-the-box approaches to infectious disease therapy including inhibition of virulence factors (e.g. S. aureus pigment) or pharmacological augmentation of phagocytic cell function (e.g. HIF-1α boosting) represent critical areas for exploration. Finally, through an NICHD-sponsored UCSD Research Program in Developmental Pharmacology, we are exploring synergy of pharmaceutical antibiotics with natural antimicrobial peptides, with a goal of optimizing therapy through innate immune sensitization. Collaborators include W. Fenical (SIO/UCSD), P. Dorrestein (UCSD), M. Burkart (UCSD) and E. Capparelli (UCSD)||Novel Antibiotic
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