George Y. Liu

Low avidity recognition of self-antigen by T cells permits escape from central tolerance.

The immunodominant epitope of myelin basic protein, Ac1-9, is encephalitogenic in H-2u mice. We have previously demonstrated that this epitope displays low affinity for I-Au and have suggested that the avidity of T cell recognition in the thymus may be compromised, enabling autoreactive T cells to escape self-tolerance. We have addressed this hypothesis directly by constructing transgenic mice expressing an encephalitogenic T cell receptor (TCR). Parenteral administration of Ac1-9 had no discernable impact on developing thymocytes. In contrast, peptide analogs displaying far higher affinity for I-Au, provoked deletion of CD4+ CD8+ cells and transient down-regulation of the TCR by mature CD4+ CD8- thymocytes. The use of analogs of intermediate affinity permitted a margin of error to be defined for the induction of tolerance and confirmed that the affinity of Ac1-9 lies well below the critical threshold.

A cholesterol biosynthesis inhibitor blocks Staphylococcus aureus virulence

Staphylococcus aureus produces hospital- and community-acquired infections, with methicillin-resistant S. aureus posing a serious public health threat. The golden carotenoid pigment of S. aureus, staphyloxanthin, promotes resistance to reactive oxygen species and host neutrophil-based killing, and early enzymatic steps in staphyloxanthin production resemble those for cholesterol biosynthesis. We determined the crystal structures of S. aureus dehydrosqualene synthase (CrtM) at 1.58 angstrom resolution, finding structural similarity to human squalene synthase (SQS). We screened nine SQS inhibitors and determined the structures of three, bound to CrtM. One, previously tested for cholesterol-lowering activity in humans, blocked staphyloxanthin biosynthesis in vitro (median inhibitory concentration ∼100 nM), resulting in colorless bacteria with increased susceptibility to killing by human blood and to innate immune clearance in a mouse infection model. This finding represents proof of principle for a virulence factor–based therapy against S. aureus.

Polymorphonuclear leukocytes mediate Staphylococcus aureus Panton-Valentine leukocidin-induced lung inflammation and injury

Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) is epidemic in the United States, even rivaling HIV/AIDS in its public health impact. The pandemic clone USA300, like other CA-MRSA strains, expresses Panton-Valentine leukocidin (PVL), a pore-forming toxin that targets polymorphonuclear leukocytes (PMNs). PVL is thought to play a key role in the pathogenesis of necrotizing pneumonia, but data from rodent infection models are inconclusive. Rodent PMNs are less susceptible than human PMNs to PVL-induced cytolysis, whereas rabbit PMNs, like those of humans, are highly susceptible to PVL-induced cytolysis. This difference in target cell susceptibility could affect results of experimental models. Therefore, we developed a rabbit model of necrotizing pneumonia to compare the virulence of a USA300 wild-type strain with that of isogenic PVL-deletion mutant and -complemented strains. PVL enhanced the capacity of USA300 to cause severe lung necrosis, pulmonary edema, alveolar hemorrhage, hemoptysis, and death, hallmark clinical features of fatal human necrotizing pneumonia. Purified PVL instilled directly into the lung caused lung inflammation and injury by recruiting and lysing PMNs, which damage the lung by releasing cytotoxic granule contents. These findings provide insights into the mechanism of PVL-induced lung injury and inflammation and demonstrate the utility of the rabbit for studying PVL-mediated pathogenesis.

Staphylococcus aureus Evades Lysozyme-Based Peptidoglycan Digestion that Links Phagocytosis, Inflammasome Activation, and IL-1β Secretion

IL-1beta produced by phagocytes is important for protection against the mucosal pathogen Staphylococcus aureus. Processing and maturation of this cytokine requires activation of the multiprotein inflammasome complex. We observed that the bacterial cell wall component peptidoglycan (PGN) must be particulate and internalized via phagocytosis to activate NLRP3 inflammasomes and IL-1beta secretion. In the context of S. aureus infection of macrophages, we find that phagocytosis and lysozyme-based bacterial cell wall degradation are necessary to induce IL-1beta secretion. Further, an S. aureus enzyme, PGN O-acetyltransferase A, previously demonstrated to make cell wall PGN resistant to lysozyme, strongly suppresses inflammasome activation and inflammation in vitro and in vivo. These observations demonstrate that phagocytosis and lysozyme-based cell wall degradation of S. aureus are functionally coupled to inflammasome activation and IL-1beta secretion and illustrate a case whereby a bacterium specifically subverts IL-1beta secretion through chemical modification of its cell wall PGN.

Color me bad: microbial pigments as virulence factors

A hallmark feature of several pathogenic microbes is the distinctive color of their colonies when propagated in the clinical laboratory. Such pigmentation comes in a variety of hues, and has often proven useful in presumptive clinical diagnosis. Recent advances in microbial pigment biochemistry and the genetic basis of pigment production have sometimes revealed a more sinister aspect to these curious materials that change the color of reflected light by selective light absorbance. In many cases, the microbial pigment contributes to disease pathogenesis by interfering with host immune clearance mechanisms or by exhibiting pro-inflammatory or cytotoxic properties. We review several examples of pigments that promote microbial virulence, including the golden staphyloxanthin of Staphylococcusaureus, the blue-green pyocyanin of Pseudomonas spp., and the dark brown or black melanin pigments of Cryptococcus neoformans and Aspergillus spp. Targeted pigment neutralisation might represent a viable concept to enhance treatment of certain difficult infectious disease conditions.

Group B streptococcal β-hemolysin/cytolysin activates neutrophil signaling pathways in brain endothelium and contributes to development of meningitis

Meningitis occurs when blood-borne pathogens cross the blood-brain barrier (BBB) in a complex interplay between endothelial cells and microbial gene products. We sought to understand the initial response of the BBB to the human meningeal pathogen group B Streptococcus (GBS) and the organisms major virulence factors, the exopolysaccharide capsule and the beta-hemolysin/cytolysin toxin (beta-h/c). Using oligonucleotide microarrays, we found that GBS infection of human brain microvascular endothelial cells (HBMEC) induced a highly specific and coordinate set of genes including IL-8, Groalpha, Grobeta, IL-6, GM-CSF, myeloid cell leukemia sequence-1 (Mcl-1), and ICAM-1, which act to orchestrate neutrophil recruitment, activation, and enhanced survival. Most strikingly, infection with a GBS strain lacking beta-h/c resulted in a marked reduction in expression of genes involved in the immune response, while the unencapsulated strain generally induced similar or greater expression levels for the same subset of genes. Cell-free bacterial supernatants containing beta-h/c activity induced IL-8 release, identifying this toxin as a principal provocative factor for BBB activation. These findings were further substantiated in vitro and in vivo. Neutrophil migration across polar HBMEC monolayers was stimulated by GBS and its beta-h/c through a process involving IL-8 and ICAM-1. In a murine model of hematogenous meningitis, mice infected with beta-h/c mutants exhibited lower mortality and decreased brain bacterial counts compared with mice infected with the corresponding WT GBS strains.

Vitamin D Induces Innate Antibacterial Responses in Human Trophoblasts via an Intracrine Pathway

Abstract The active form of vitamin D, 1,25-dihydroxyvitamin D (1,25(OH)2D), is a potent inducer of the antimicrobial protein cathelicidin, CAMP (LL37). In macrophages this response is dependent on intracrine synthesis of 1,25(OH)2D from precursor 25-hydroxyvitamin D (25OHD), catalyzed by the enzyme 25-hydroxyvitamin D-1alpha-hydroxylase (CYP27B1). In view of the fact that trophoblastic cells also express abundant CYP27B1, we postulated a similar intracrine pathway for induction of CAMP in the placenta. Analysis of placenta explants, primary cultures of human trophoblast, and the 3A trophoblastic cell line treated with 1,25(OH)2D (1–100 nM) revealed dose-dependent induction of CAMP similar to that observed with primary cultures of human macrophages. Also consistent with macrophages, induction of trophoblastic CAMP was enhanced via intracrine conversion of 25OHD to 1,25(OH)2D. However, in contrast to macrophages, induction of CAMP by vitamin D in trophoblasts was not enhanced by costimulation with Toll-like receptor ligands, such as lipopolysaccharide. Despite this, exposure to vitamin D metabolites significantly enhanced antibacterial responses in trophoblastic cells: 3A cells infected with Escherichia coli showed decreased numbers of bacterial colony-forming units compared with vehicle-treated controls when treated with 25OHD (49.6% ± 10.9%) or 1,25(OH)2D (45.4% ± 9.2%), both P < 0.001. Treatment with 25OHD (1–100 nM) or 1,25(OH)2D (0.1–10 nM) also protected 3A cells against cell death following infection with E. coli (13.6%–26.9% and 22.3%–40.2% protection, respectively). These observations indicate that 1,25(OH)2D can function as an intracrine regulator of CAMP in trophoblasts, and may thus provide a novel mechanism for activation of innate immune responses in the placenta.

Carotenoid-Related Alteration of Cell Membrane Fluidity Impacts Staphylococcus aureus Susceptibility to Host Defense Peptides

ABSTRACT Carotenoid pigments of Staphylococcus aureus provide integrity to its cell membrane (CM) and limit oxidative host defense mechanisms. However, the role of carotenoids in staphylococcal resistance to nonoxidative host defenses has not been characterized. The current study examined the relationship among CM carotenoid content, membrane order, and in vitro susceptibility to daptomycin or to prototypic neutrophil-derived, platelet-derived, or bacterium-derived cationic antimicrobial peptides (human neutrophil defensin-1 [hNP-1], platelet microbicidal proteins [PMPs], or polymyxin B, respectively). A previously characterized methicillin-susceptible Staphylococcus aureus (MSSA) isogenic clinical strain set was used, including a parental isolate with an intact carotenoid biosynthetic operon (crtOPQMN) containing the crtM gene encoding early steps in staphyloxanthin biosynthesis, a crtM deletion mutant, and a crtMN multicopy plasmid-complemented variant. Compared to the parental and crtM knockout strains, the crtMN-complemented strain exhibited (i) increased carotenoid production, (ii) increased CM rigidity (P < 0.001), and (iii) uniformly reduced susceptibility to killing by the above-mentioned range of cationic peptides (statistically significant for hNP-1 [20 μg/ml]; P = 0.0037). There were no significant differences in phospholipid composition and asymmetry, fatty acid profiles, surface charge, or cell wall thickness among the strain set. Collectively, these data support the concept that carotenoid biosynthesis can contribute to the ability of S. aureus to subvert nonoxidative host defenses mediated by cationic peptides, potentially by increasing target membrane rigidity.

Staphylococcus aureus Panton-Valentine leukocidin contributes to inflammation and muscle tissue injury.

Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) threatens public health worldwide, and epidemiologic data suggest that the Panton-Valentine Leukocidin (PVL) expressed by most CA-MRSA strains could contribute to severe human infections, particularly in young and immunocompetent hosts. PVL is proposed to induce cytolysis or apoptosis of phagocytes. However, recent comparisons of isogenic CA-MRSA strains with or without PVL have revealed no differences in human PMN cytolytic activity. Furthermore, many of the mouse studies performed to date have failed to demonstrate a virulence role for PVL, thereby provoking the question: does PVL have a mechanistic role in human infection? In this report, we evaluated the contribution of PVL to severe skin and soft tissue infection. We generated PVL mutants in CA-MRSA strains isolated from patients with necrotizing fasciitis and used these tools to evaluate the pathogenic role of PVL in vivo. In a model of necrotizing soft tissue infection, we found PVL caused significant damage of muscle but not the skin. Muscle injury was linked to induction of pro-inflammatory chemokines KC, MIP-2, and RANTES, and recruitment of neutrophils. Tissue damage was most prominent in young mice and in those strains of mice that more effectively cleared S. aureus, and was not significant in older mice and mouse strains that had a more limited immune response to the pathogen. PVL mediated injury could be blocked by pretreatment with anti-PVL antibodies. Our data provide new insights into CA-MRSA pathogenesis, epidemiology and therapeutics. PVL could contribute to the increased incidence of myositis in CA-MRSA infection, and the toxin could mediate tissue injury by mechanisms other than direct killing of phagocytes.

A group B streptococcal pilus protein promotes phagocyte resistance and systemic virulence

Group B Streptococcus (GBS) is a major cause of invasive bacterial infections in newborns and certain adult populations. Surface filamentous appendages known as pili have been recently identified in GBS. However, little is known about the role of these structures in disease pathogenesis. In this study we sought to probe potential functional role(s) of PilB, the major GBS pilus protein subunit, by coupling analysis of an isogenic GBS pilB knockout strain with heterologous expression of the pilB gene in the nonpathogenic bacterium Lactococcus lactis. We found the knockout GBS strain that lacked PilB was more susceptible than wild‐type (WT) GBS to killing by isolated macrophages and neutrophils. Survival was linked to the ability of PilB to mediate GBS resistance to cathelicidin antimicrobial peptides. Furthermore, the PilB‐deficient GBS mutant was more readily cleared from the mouse bloodstream and less‐virulent in vivo compared to the WT parent strain. Strikingly, overexpression of the pilB gene alone in L. lactis enhanced resistance to phagocyte killing, increased bloodstream survival, and conferred virulence in a mouse challenge model. Together these data demonstrate that the pilus backbone subunit, PilB, plays an integral role in GBS virulence and suggests a novel role for gram‐positive pili in thwarting the innate defenses of phagocyte killing.— Maisey H.C., Quach, A., Hensler, M. E., Liu, G. Y., Gallo, R. L., Nizet, V., Doran K. S. A group B streptococcal pilus protein promotes phagocyte resistance and systemic virulence. FASEB J. 22, 1715–1724 (2008)