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| Nataliya Balashova, PhD, research associate, and her mentor, Scott Kachlany, PhD, associate professor, Oral Biology Department, UMDNJ-New Jersey Dental School |
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Microbial pathogenicity refers to the disease-causing activity of microorganisms. Bacterial toxins play an important role in the development of bacterial infection. HACEK organisms (Haemophilus species, Aggregatibacter actinomycetemcomitans, Cardiobacterium hominis, Eikenella corrodens, and Kingella species) are a group of gram-negative bacteria that normally reside in the oral cavity and share an enhanced capacity to produce infective endocarditis and other systemic infections. In our lab, we focus on identification and characterization of bacterial toxins from A. actinomycetemcomitans and K. kingae.
A variety of bacteria live in the human body. The oral cavity can act as the site of origin for dissemination of pathogenic organisms to distant body sites. Recent work indicates that certain bacteria normally found in the oral cavity can cause systemic infection. This finding is having an impact on the study of the virulence mechanisms of these pathogens, in particular the mechanisms of their defenses against inflammatory bactericidal mechanisms by the host.
Pathogenicity in bacteria may be associated with unique structural components of the cells, e.g. capsules, pili, lipopolysaccharides or active secretion of substances such as toxins that either damage host tissues or protect the bacteria against host defenses. Toxic substances produced by bacteria may be transported by the vascular or lymphatic system and cause cytotoxic effects at tissue sites remote from the original point of invasion or growth.
HACEK organisms share an enhanced capacity to produce various infections, including infective endocarditis (IE), a severe infection of heart valves. These organisms are responsible for 5-10% of cases of IE involving native valves and are the most common cause of gram-negative endocarditis among persons who do not abuse intravenous drugs. Sixty percent of HACEK IE cases are associated with various types of dental pathology. In addition to valvular infections, they also produce: bacteremias; various types of abscesses; peritonitis; otitis media; conjunctivitis; pneumonia; septic arthritis; osteomyelitis; urinary tract, wound, and periodontal infections; and brain abscesses. Under laboratory conditions these bacteria are slow growers, have fastidious culturing requirements and prefer a carbon dioxide-enriched atmosphere. My research is focused on the study of toxins produced by two HACEK bacteria.
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Figure 1. A model describing the interplay between LtxA and Cu, Zn SOD of A. actinomycetemcomitans (Aa) produces LtxA and Cu, Zn SOD. They interact presumably on the outer membrane. Cu, Zn SOD inactivates superoxide generated by host white blood cells (WBC). LtxA attacks WBC and red blood cells (RBC). Oxidation of hemoglobin released from RBC causes heme accumulation in the environment. Heme may bind to Cu, Zn SOD and serve as a source of iron and heme for Aa. LtxA production, hemolytic activity and efficiency of heme binding by Cu, Zn SOD may be regulated by free iron or heme in the environment. |
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Figure 2. Trypan blue assay after four hours of HL-60 cells (human leukemia cell line) treatment with K. kingae membrane fraction. Dark circles indicate dead cells. |
Aggregatibacter (Actinobacillus) actinomycetemcomitans is an oral pathogen that is the etiologic agent of localized aggressive periodontitis (LAP). As one of its virulence factors, A. actinomycetemcomitans produces leukotoxin (LtxA), which is a member of the RTX toxin (repeats in toxin) family of secreted bacterial toxins and is known to lyse human leukocytes. We demonstrated that in addition to leukotoxic activity, A. actinomycetemcomitans LtxA also has the ability to lyse erythrocytes. While growing A. actinomycetemcomitans strains isolated from patients with LAP and IE on Columbia agar with 5% sheep blood, we noticed a b-hemolytic reaction (clear halo around colonies) by leukotoxic strains but not their ltxA mutants. In our lab we designed a procedure for LtxA purification from the cell culture supernatant.
Iron is essential for the growth and survival of bacterial cells. Hemoglobin and heme, released from erythrocytes, are potential sources of iron for A. actinomycetemcomitans. We found that b-hemolysis was suppressed under iron-excess, and enhanced under iron-limiting, growth conditions. The expression of ltxA and ltxA-associated genes was not regulated by iron, but secretion of LtxA was completely inhibited by free iron. Thus, iron regulates LtxA secretion in A. actinomycetemcomitans in a manner independent of gene regulation.
We have recently detected that Cu, Zn superoxide dismutase (Cu, Zn SOD) from A. actinomycetemcomitans interacts with LtxA. Bacterial Cu, Zn SODs have a role in protecting pathogens from oxygen radicals released from host inflammatory cells. Cu, Zn SOD also has been shown to bind heme and potentially play a role in iron and heme acquisition. A. actinomycetemcomitans Cu, Zn SOD mutants displayed decreased survival in the presence of reactive oxygen and nitrogen species (ROS and RNS). We found that LtxA was very sensitive to ROS and RNS, and Cu, Zn SOD protected the protein from damage. Therefore, we suggest that interaction between LtxA and Cu, Zn SOD may result in protection of LtxA once it is secreted (Fig. 1).
Thus, our results show a novel additional function of the toxin and suggest its role in iron acquisition. In light of these results, the diagnostic criteria for clinical identification of A. actinomycetemcomitans should be re-evaluated. We propose that A. actinomycetemcomitans hemolysis is an important property for the bacterium to cause or sustain disease. When it becomes systemic and infects heart tissue, A. actinomycetemcomitans may use LtxA to destroy erythrocytes. Cu, Zn SOD from A. actinomycetemcomitans may play a role in protecting bacteria and heme acquisition. Our future studies seek to understand the mechanisms of hemolysis and iron uptake in A. actinomycetemcomitans.
Another HACEK member I am concentrating on is Kingella kingae, a coccobacillus of the Nesseriaceae family. Recently, as the result of improved isolation and identification techniques, an increasing number of invasive K. kingae infections have been reported throughout the world, suggesting that the organism is an important cause of bacteremia and septic arthritis in pediatric patients. Unlike other HACEK organisms, IE caused by this organism progresses quite rapidly. Despite the emerging body of information on the clinical and diagnostic aspects of K. kingae infections, the virulence mechanisms of this pathogen remain largely unknown.
In an attempt to identify virulence factors, we tested K. kingae cells and cell culture supernatant toxicity on different human cells. The hemolytic activity was found in K. kingae cell culture supernatant. The hemolysin was purified and identified as an RTX toxin. In contrast, K. kingae cells could not lyse erythrocytes and epithelial cells efficiently, but were highly toxic to human white blood cells, including monocytes, myeloblasts and megakaryoblasts. K. kingae cell fractionation revealed that the leukotoxic factor (Ktx) is membrane-associated (Fig. 2). Protease and high temperature treatment abolished toxic activity of the K. kingae membrane fraction indicating that Ktx is proteinaceous.
Our results suggest that K. kingae produces at least two different toxins: the RTX hemolysin that is secreted into cell culture supernatant and leukotoxic Ktx that is associated with cell membranes. This is the first evidence of a leukotoxin in K. kingae that may be an important virulence factor for evasion of the host immune response during infection. The experiments on the Ktx identification are underway and I believe it will become the major focus of my future independent research.
Nataliya Balashova received her PhD from the Institute of Biochemistry and Physiology of Microorganisms, Pushchino, Russia, in 2000. She did her first postdoctoral training in the Department of Pharmacology and Physiology at UMDNJ-New Jersey Medical School. In 2004, she joined the Oral Biology Department of UMDNJ-New Jersey Dental School and is currently a research associate there. Her research is supported by an NIH-funded fellowship.
