Victor Nizet

Affiliation: UCSD SOM

Associate Professor of Pediatrics

Biography

Victor Nizet, M.D. is an Associate Professor of Pediatrics in the Division of Infectious Diseases at the University of California, San Diego School of Medicine. Dr. Nizet received his medical training at Stanford University School of Medicine, completed a Residency and Chief Residency in Pediatrics at Harvard University's Children's Hospital in Boston, and a Fellowship in Pediatric Infectious Diseases at the University of Washington in Seattle. In 1997, Dr. Nizet joined the faculty at UCSD and directs basic research program focusing on the molecular genetics and pathogenesis of human streptococcal infections and the innate immune system. In addition, he serves as an attending physician on the Pediatric Infectious Diseases consult services at Children's Hospital & Health Center, UCSD Medical Center, and other affiliated San Diego area hospitals. In 2002, Dr. Nizet was the recipient of the Pediatric Infectious Diseases Society Young Investigator Award and was named the 39th Edward Mallinckrodt, Jr. Foundation Scholar.

Research Summary

Our laboratory interest is in bacterial pathogenesis and the innate immune system, with a special focus on human streptococcal infections. Using a molecular genetic approach, we seek to discover and characterize streptococcal virulence determinants involved in cytotoxicity, adherence, invasion, inflammation and resistance to immunologic clearance. Specific projects include (1) identification of novel GAS factors involved in host cell attachment and invasion, (2) the genetic basis of Group B Streptococcus (GBS) and GAS (streptolysin S) hemolysin production and the roles of these cytotoxins in producing host cell injury and immune activation, (3) the mechanisms by which GBS penetrate the human blood-brain barrier to produce meningitis, and (4) bacterial genetic approaches to understanding the role of antimicrobial peptides in innate host mucosal defense.

Group A Streptolysin:

The principal factor responsible for Β-hemolysis in GAS is streptolysin S (SLS), an oxygen-stable, non-immunogenic, broad-spectrum cytolysin that has yet to be fully purified. Insertion of SLS into the RBC membrane results in transmembrane pore formation and osmotic cell lysis. Our research has applied a molecular approach to discover the genetic basis of SLS production and the role of this potent exotoxin in disease pathogenesis. These studies are the product of a rewarding and ongoing collaboration with the laboratories of Joyce DeAzavedo at Mt. Sinai Hospital in Toronto and Bernie Beall at athe Centers for Disease Control in Atlanta.

A locus of 9 contiguous ORFs associated with SLS production was identified by analyzing random transposon mutants of GAS exhibiting a nonhemolytic phenotype. This locus, conserved among GAS of various emm genotypes, has been named "sag" for "streptolysin-associated genes" (Figure 1)

The sag locus has many features characteristic of a bacteriocin biosynthetic operon. The first gene, sagA encodes a 53 aa candidate prepropeptide. Within SagA is a typical Gly-Gly cleavage motif separating an N-terminal 23 aa leader from a 30 aa propeptide matching the calculated size of mature SLS (2.9 kD). The propeptide is highly enriched in amino acids (Ser, Thr, Gly, Cys) that are the precursors for post-translational modification and thioether bond formation in other cyclical bacteriocin toxins. The sagG-sagI genes have strong homology to ATP-binding cassette (ABC) transporters commonly required for the export of bacteriocins peptides. The SagB and SagE predictedproteins share very weak homology to a bacteriocin modifying enzyme and immunity protein, respectively; the other sag gene products have no significant Genbank homologies. RT-PCR analysis confirms an operon structure, as the sagB-sag I genes utilize the same promoter as sag A. As in other bacteriocin operons, a “leaky” terminator situated between sagA and sag B acts as a regulatory mechanism yielding an abundance of structural gene transcript ( sag A alone) and smaller amounts of mRNA for downstream genes involved in modification, processing and export of the mature toxin. Plasmid integrational mutagenesis verified the transposon mutant phenotypes and defined the functional boundaries of the sag operon; targeted integrations in each gene yielded nonhemolytic GAS, while mutations upstream of the sag promoter or downstream of sagI did not affect SLS production. Cloning of the entire 9-gene sag locus in nonhemolytic Lactococcus lactis resulted in robust and stable Β-hemolytic transformants. These experiments demonstrated the intact sag locus is both necessary and sufficient for SLS production (Figure 2).

Homologues of the GAS sag operon for SLS biosynthesis have recently been identified in invasive human isolates of Β-hemolytic group C and G streptococci and the fish pathogen S. iniae. The contribution of SLS to the pathogenesis of streptococcal necrotizing soft tissue infection has been examined in the murine model of necrotizing fasciitis. In this model, wild-type bacteria elicit a ulcer with bacterial proliferation, neutrophilic inflammation, and histopathologic evidence of vascular injury and tissue necrosis. In contrast to the parent strains, isogenic SLS-negative sag gene mutants do not develop ulcers, and biopsy of the inoculation site demonstrates bacterial clearance and minimal degrees of inflammation or tissue injury (Figure 3). In vitro studies suggest that SLS can contribute to pathogenesis both by direct cytotoxicity and by inhibiting neutrophil phagocytosis. The latter may help explain the paradox of decreased bacterial clearance despite the intense neutrophil influx seen with the wild-type bacteria.

Our ongoing collaborative research takes advantage of the unique genetic information and specific bacteriologic reagents we have generated to further study the basic biology and pathogenic role of the SLS toxin. Efforts include studies to (1) purify the toxin and understand its biosynthetic pathway, (2) determine the specific SagA amino acid residues critical for its cytolytic action, (3) characterize SLS antiphagocytic, proinflammatory and antibacterial properties, (4) assess the contribution of the toxin to disease pathogenesis in vivo, and (5) determine the potential benefits of SLS neutralization in the treatment of invasive infection.

Group B Streptolysin Project:

GBS are the leading cause of meningitis in human newborns. While all serotypes may produce meningitis, capsule serotype III strains account for ~80% of cases. Although neonatal meningitis develops as a consequence of hematogenous spread of the organism, the factors responsible for GBS entry into the central nervous system (CNS) have not been determined. The blood-brain barrier (BBB) consists of a single layer of specialized brain microvascular endothelial cells (BMEC) which exhibit continuous tight junctions and are responsible for maintaining biochemical homeostasis within the CNS. We had observed GBS organisms inside the BMEC of neonatal rats infected intraperitoneally and sacrificed histopathologic examination of brain tissue. We hypothesized that GBS produce meningitis because of a unique capacity to invade human brain microvascular endothelial cells. To test this hypothesis, we developed together with Prof. Kwang Sik Kim (now at Johns Hopkins) an in vitro model of GBS blood-brain barrier interactions using BMEC isolated from a human, immortalized by SV40 transformation, and propagated in tissue culture monolayers. GBS invasion of BMEC was demonstrated by electron microscopy, the first ever photographic evidence that a human meningitis pathogen could penetrate the cells which constitute the blood-brain barrier. Intracellular GBS were found within membrane-bound vacuoles, suggest the organism induced its own endocytotic uptake.

We adapted a gentamicin protection assay to quantify intracellular GBS. Serotype III strains, which account for the majority of CNS isolates, invaded BMEC more efficiently that strains from other serotypes. GBS invasion required active bacterial DNA, RNA and protein synthesis, as well as microfilament and microtubule elements of the eukaryotic cytoskeleton. At high bacterial densities, GBS invasion of BMEC was accompanied by evidence of cellular injury, which in turn was correlated to hemolysin/cytolysin production by the organism. Finally, we demonstrated GBS transcytosis across intact, polar BMEC monolayers grown on Transwell membranes. We concluded that GBS invasion of BMEC is a primary step in the pathogenesis of neonatal meningitis, allowing bacteria access to the CNS by transcytosis or by injury and disruption of the endothelial BBB.

Ongoing studies led by our colleague Dr. Kelly Doran employ genetic techniques including transposon mutagenesis and heterologous expression to identify specific GBS genes and gene products responsible for GBS invasion of BMEC. We have also utilized high density oligonucleotide microarrays to identify host genes important in the initial inflammatory response of the BBB to a bacterial pathogen. We found that GBS infection induced a highly specific and coordinate set of genes known to orchestrate neutrophil recruitment, activation and enhanced survival. Prominent induced genes included the C-X-C family chemokines interleukin (IL)-8, Gro-alpha and Gro-beta, along with IL-6, granulocyte-macrophage colony stimulating factor (GM-CSF), myeloid cell leukemia sequence 1 (Mcl-1) and intercellular adhesion molecule 1 (ICAM-1). Key findings were confirmed by Real Time RT-PCR and immunoassays. Specific bacterial triggers for BBB gene activation were sought by parallel studies using isogenic mutants lacking important GBS virulence factors. AΒ-h/c deficient strain exhibited a significantly reduced ability to induce the same subset of genes, identifying it as the principal provocative factor for BBB activation. In contrast, an unencapsulated strain induced greater expression of several genes, suggesting the capsule may act as a “cloak” to diminish host recognition of the pathogen. It appears the innate immune response of BBB endothelium to GBS is to summon circulating neutrophils, a response that is modulated by specific bacterial virulence determinants.

Antimicrobial peptide project:

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 antimicrobial peptides (AMPs), a protection strategy conserved from insects through man. In mammals, gene families encoding AMPs include defensins and cathelicidins. Our AMP research efforts are the product of a fruitful collaboration with Richard Gallo, a UCSD dermatologist and investigator whose laboratory studies the cellular biology and immunology of the skin. The Gallo laboratory has made several important advances in our understanding of the cathelicidin family of antimicrobial peptides, including the first purification of the porcine cathelicidin PR-39 and the discovery of the murine cathelicidin CRAMP. Our collaboration adopts a combined mammalian and bacterial genetic approach to eludating the contributions of cathelicidins to host immunity.

Humans and mice each express a single cathelicidin, which are encoded by similar genes and have similar alpha-helical structures, spectra of antimicrobial activity and tissue distribution. Cathelicidins effectively kill group A Streptococcus (GAS) in vitro, and cathelicidin production greatly is increased in the skin after infectious challenge with GAS. To assess directly the function of cathelicidins in vivo, the Gallo laboratory generated mice that are null for CRAMP by targeted recombination. When challenged with GAS sucutaneously, CRAMP-deficient mice developed much larger areas of necrotic infection the wild-type mice and failed to clear the replicating bacteria from the wound. To corroborate this finding, we identified a GAS transposon mutant with increased resistance to cathelicidin killing in vitro. The transposon insertion mapped to a protein with features of a transcriptional regulator and was confirmed by targeted plasmid integrational mutagenesis. Mice infected with this mutant developed lesions of larger size and longer duration than those infected with the cathelicidin-sensitive GAS parent strain. In effect, induction of cathelicidin resistance in the bacterial pathogen reproduced the phenotype of the CRAMP-knockout mouse. The pattern of findings in the skin were mirrored in the results of whole blood killing assays. These studies represented the first in vivo demonstration that endogenous expression of a mammalian antimicrobial peptide provides defense against an invasive bacterial infection.

Ongoing collaborative projects with the Gallo laboratory explore additional biological functions of the cathelicidin molecule and its regulation in response to infectious challenge, the molecular genetic and phenotypic basis of bacterial sensitivity or resistance to cathelicidin action, and the impact of bacterial cathelicidin resistance on the epidemiology ofinfectious diseases.

References

References From PubMed (NCBI)